Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1

Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N-acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function.     

Immunohistochemical localization of oviductin in the endometrial lining of the golden hamster (Mesocricetus auratus) during the estrous cycle and early gestation

Summary Oviductal non-ciliated secretory epithelial cells, under hormonal stimulation, synthesize and secrete a family of glycoproteins referred to as oviductins. These glycoproteins are found in oviductal fluid in several mammalian species, and have been localized in the oviduct, and in the zona pellucida of ovulated oocytes. In the golden hamster, this glycoprotein is named hamster oviductin-1. Recently, an immunofluorescent study on hamster uterine tissue has revealed the presence of the glycoprotein in luminal epithelial cells in a heterogeneous labelling pattern during the estrous cycle. The mechanism of endometrial epithelial cell receptivity to hamster oviductin-1 is not known.In this study, immunohistochemical studies were performed using a monoclonal antibody against the oviductin in conjunction with silver enhancement technique, in an attempt to determine further the factors playing a role in uterine receptivity to oviductin-1. Paraffin sections of hamster uterus obtained from different stages of the estrous cycle and from days 1–6 of gestation, and paraffin sections of hamster oviduct obtained from days 1–6 of gestation were used in this study. The results we obtained using the silver enhancement technique show that hamster uterus luminal epithelial cells exhibit a homogeneous, high intensity immunolabelling pattern throughout the estrous cycle, whereas, during gestation, labelling intensity decreases as the period for blastocyst implantation approaches. Oviduct epithelial cells revealed no definite fluctuating pattern in immunolabelling intensities during gestation, indicating no change in synthesis and secretion of the glycoprotein during this period.It is speculated that receptors for hamster oviductin-1 are present at the apical cell surface of endometrial cells and that implantation of the developing blastocyst into the uterine wall is possible only following downregulation of these receptors.The use of the silver enhancement technique proves to be an effective tool in immunohistochemical studies at the light microscope level, as seen through this study.     

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells.

The glycosyltransferase core 2 beta1-6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8(+) T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8(+) cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.     

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.     

Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.     

Antioxidant defenses are modulated in the cow oviduct during the estrous cycle

The balanced presence of reactive oxygen species and antioxidants has a positive impact on sperm functions, oocyte maturation, fertilization, and embryo development in vitro. The mammalian oviduct is likely to provide an optimal environment for final gamete maturation, sperm-egg fusion, and early embryonic development. However, the expression and distribution of antioxidant enzymes in the bovine oviduct are poorly characterized. We analyzed the mRNA expression and enzymatic activities of major antioxidants glutathione peroxidase (GPx), superoxide dismutase (Cu,ZnSOD), and catalase in the bovine oviduct throughout the estrous cycle. The high levels of expression for GPx-3 in the isthmus were in contrast to expression of GPx-1 and GPx-2, which occurred mostly in the ampulla and infundibulum of the oviduct. The highest levels of mRNA expression for GPx-1 were observed toward the end of the estrous cycle before ovulation, whereas GPx-2 was mostly expressed at midcycle. Catalase and Cu,ZnSOD mRNA analyses revealed a homogenous expression along the oviduct. The highest levels of glutathione and enzymatic activities for GPx and catalase occurred at the middle (10-12 days) and end (18-20 days) of the estrous cycle, whereas total SOD activity remained constant throughout the estrous cycle in the oviductal fluids. These findings underscore the importance of hydrogen peroxide and hydroperoxide removal by GPx in the oviduct. The heterogeneous expression of antioxidants such as GPx along the oviduct is a possible indication of their physiological role in the events leading to successful fertilization and implantation in vivo.     

Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Distribution of lectin-binding glycosidic residues in the hamster follicular oocytes and their modifications in the zona pellucida after ovulation.

In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohydrate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin-gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progressively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB(4) was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analysis was also performed on labeling by lectin-gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization.     

Oviductal progesterone concentration and its spatial distribution in cyclic and early pregnant cows

Changes and local distribution of oviductal progesterone (P(4)) concentration during the estrous cycle and early pregnancy in cows were investigated. Intact reproductive tracts were collected from 16 Holstein cows at an abattoir. Samples were classified in to 4 stages (follicular, postovulatory, luteal and early pregnant,< 20 d) based on visual observation of corpus luteum (CL), uterine characteristics and luteal P(4) concentrations. Oviducts were separated from the uterus at the utero-tubal junction and divided into 4 parts: fimbriae, proximal, medial and distal parts. Luteal tissue samples were also collected. Progesterone levels in oviductal and luteal tissues were determined by radioimmunoassay (RIA). Comparatively higher (P < 0.001) P(4) levels were found in stages with a functioning CL ( luteal phase and early pregnancy) than in those with a regressing CL (follicular phase and post ovulation). The oviduct ipsilateral to the CL bearing ovary during the luteal phase and early pregnancy showed higher ( P < 0.001) P(4) concentrations than the contralateral side. Such a difference was not observed during the follicular phase or post ovulation. The ipsilateral oviduct to the functioning CL at early pregnancy showed higher (P <0.05) P(4) levels than at the luteal phase, while no significant difference in luteal P(4) levels between these 2 stages was observed. Neither were any differences in P(4) concentration within the oviduct observed during any phase of the estrous cycle or during early pregnancy. A positive relationship between luteal and oviductal P(4) concentrations was noted. In conclusion, changes in P(4) levels in the oviduct depend on the location and functional stage of the CL. Localized levels of P(4) in the oviduct may be due to local delivery of P(4) from the CL.     

Differential distribution of lectin-binding glycoconjugates in the secretory granules of hamster oviductal ampulla during the estrous cycle: a quantitative cytochemical analysis

 High resolution lectin-gold cytochemistry was used to quantitatively analyze the distribution of glycoconjugates in the hamster oviductal ampulla during the five stages of the estrous cycle. Lectins binding to N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates in the secretory granules of ampullary epithelial secretory cells showed staining of equal intensity throughout the five different stages of the estrous cycle. In contrast, the labeling intensity of glycoconjugates which contain N-acetylglucosamine as terminal sugar residues reached its maximum around the time of ovulation, i.e., at proestrus. Glycoconjugates which carry fucose and mannose as terminal sugar residues appeared to be totally absent from the secretory granules of the oviductal ampulla during the estrous cycle. Together, electron microscopic observations combined with quantitative results indicate that N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates may be secreted into the ampullary lumen irrespective of the stage of the estrous cycle, whereas the secretion of certain N-acetylglucosamine-associated glycoconjugates is stage specific and reaches its peak at the time of ovulation. These findings suggest that, at the time of ovulation, the ampullary epithelium changes its secretory activity and contributes its secretory products to the zona pellucida of oocytes freshly released from the ovary. Accepted: 10 August 1998     

Histological changes and transglutaminase 2 expression in the oviduct of advanced pregnant cows

The oviduct is a dynamic organ that has not been assigned specific functions during advanced pregnancy. However, since changes in the oviductal epithelium during the estrous cycle are attributed mainly to variations in estradiol (E2) levels, and E2 levels increase along pregnancy, we hypothesized that advanced pregnant cows should present changes in the oviductal epithelium. In advanced pregnant cows, the oviducts showed higher leaf-like folds and lower mucosa width and epithelium height than those of cycling animals. Also, PAS-positive apical protrusions and TUNEL-positive extruded cytoplasmic material were observed in advanced pregnant cows. Oviductal fluid from advanced pregnant cows showed lower protein concentration than that from cycling cows. Transglutaminase 2 (TG2) was detected exclusively in oviductal fluid of pregnant cows but not in cells from any stage, whereas its mRNA was detected in different amounts in cells from all stages. This protein was identified by LC/MS-MS and its identity was corroborated by Western blot. The observations in histology of the epithelium and the presence of TG2 in oviductal fluid correlate with high levels of E2 in serum. In conclusion, important histological changes in the oviductal epithelium and secretion of TG2 to the oviductal fluid appear to be triggered by the high E2 levels exclusive of advanced pregnancy.     

Modification of CD43 and other lymphocyte O-glycoproteins by core 2 N-acetylglucosaminyltransferase

CD43, the major leukocyte sialoglycoprotein, is expressed onT lymphocytes in two predominant glycoforms. CD43 115 kDa isa pan T cell marker and is specifically recognized by the monoclonalantibody S7. CD43 130 kDa is associated with T cell activationand is specifically recognized by the monoclonal antibody 1B11.The thymoma EL-4 has been identified to express mainly CD43115 kDa and little or no CD43 130 kDa. Transfection of EL-4cells with core 2 ß16N-acetylgIucosaminyltransferase(C2GnT), an enzyme in the O-glycan biosynthesis pathway, resultedin an enhanced expression of the 1B11 epitope, CD43 130 kDa,and a loss of expression of the S7 epitope, CD43 115 kDa. Analysisof CD43 by SDS-PAGE revealed that CD43 in C2GnT transfectedEL-4 cells has a molecular weight of 125 kDa compared to 115kDa in nontransfected or control transfected EL-4 cells. SDS-PAGEanalysis of three other lymphocyte O-glycoproteins, CD44, CD45,and RPTPa., revealed that C2GnT expression resulted in a molecularweight increase of approximately 3–5 kDa for each of thesethree cell surface glycoproteins. Our data indicate that, whileCD43 may be a predominant substrate for C2GnT, other lymphocyteO-glycoproteins are also modified by this glycosyltransferase.Increased reactivity of cells with the monoclonal antibody 1B11,which specifically detects the expression of murine CD43 130kDa, may thus be a marker of increases in branching of O-linkedglycans generally. CD43 core 2 N-acetylglucosaminyltransferase lymphocyte glycoproteins     

De novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture

The objective of the present study was to identify and characterize in a limited manner the major de novo oviductal secretory proteins (OSP) synthesized and released by the porcine oviduct. Oviductal tissue was collected on various days of the estrous cycle (EC) and early pregnancy (EP) and cultured in a modified minimal essential medium supplemented with 100 muCi L-[3H]-leucine. Oviductal secretory activity, as measured by the rate of incorporation of 3H-leucine (dpm/mg wet tissue weight) into nondialyzable macromolecules, was greatest (P less than .01) between days 0 and 2 and reached its lowest levels on days 10 to 15. There was no difference between left and right side or pregnancy status. This increased rate of incorporation at proestrus and estrus is temporally associated with elevated levels of estrogen. Incorporation rate for ampulla was greater than for the isthmus. Analysis of oviductal culture medium by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography revealed three protein bands of relative molecular weight (Mr) 335,000, 115,000, and 85,000, which were associated with proestrus, estrus, and metestrus and were not detectable on other days. All three proteins also incorporated 3H-glucosamine. The 115,000 Mr band was the major 3H-glucosamine-labeled protein. Two protein bands (Mr 60,000 and 20,000) were expressed with increasing progesterone during diestrus. Other de novo synthesized protein bands appear to be present throughout the EC and EP with little modulation by estrogen or progesterone. Thus, this study demonstrates that for the porcine oviduct, the increase in the incorporation rate of 3H-leucine into OSP by both whole oviduct and ampulla and de novo synthesis and secretion of three glycoproteins, Mr 335,000, 115,000, and 85,000, were associated with proestrus and estrus when events such as fertilization and early cleavage stages of embryo development occurred.     

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.