Gastrointestinal mucins of Fut2-null mice lack terminal fucosylation without affecting colonization by Candida albicans

Posttranslational modification of apomucins by the sequential action of glycosyltransferases is required to produce mature mucins. The Secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (EC 2.4.1.69) that catalyzes addition of terminal alpha(1,2)fucose residues on mucins and other molecules in mucosal epithelium. Mutant mice containing targeted replacement of Fut2 with the bacterial reporter gene lacZ were studied to determine the affect of the loss of Fut2 on glycosylation of mucins in the gastrointestinal tract. By whole organ X-gal staining, lacZ activity is prominently expressed in the foveolar pit and chief cells of the glandular stomach, Brunner's glands of the duodenum, and goblet cells in the large intestine of Fut2-LacZ-null mice. Staining with Aleuria aurantia agglutinin demonstrates loss of L-fucosylated epithelial glycans throughout the gastrointestinal tract of Fut2-LacZ-null mice, however, histologic appearance of the tissues appears normal. Analysis of oligosaccharides released from insoluble colonic mucins, largely Muc2, by mass spectrometry shows complete lack of terminal fucosylation of O-linked oligosaccharides in Fut2-LacZ-null mice. Precursor glycans accumulate with no evidence of compensation by other fucosyltransferases or sialyltransferases on mucin glycosylation. Because Candida albicans has been reported to adhere to intestinal mucins creating a potential reservoir associated with vaginitis, Fut2-LacZ-null and wild-type mice were inoculated by gastric lavage with C. albicans. We observe no difference in colonization between genotypes suggesting mucin terminal fucosylation does not significantly influence C. albicans-host interaction in the intestine, highlighting that infections caused by the same organism at different mucosal surfaces are not equal.     

Detection of bradykinin and bradykinin-beta(2) receptors in the porcine endometrium during the estrous cycle and early pregnancy

During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin beta(2) receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12-18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12-18 of pregnancy compared with the estrous cycle. Endometrial bradykinin beta(2) receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin beta(2) receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.     

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

Disruption of the tissue inhibitor of metalloproteinase-1 gene results in altered reproductive cyclicity and uterine morphology in reproductive-age female mice

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.     

Acute and chronic estrogen supplementation decreases uterine sympathetic innervation in ovariectomized adult virgin rats

Uterine innervation undergoes substantial reorganization associated with changes in reproductive status. Nerves innervating the uterus are decreased in pregnancy and puberty, and even the normal rodent estrous cycle is characterized by fluctuations in numbers of myometrial nerve fibers. During the follicular (proestrus/estrous) phase of the estrous cycle, intact nerves are rapidly depleted and then return over the next 2-3 days in the luteal (metestrus/diestrus) phase. We hypothesize that uterine nerve depletion is initiated by increased circulating estrogen in the follicular phase. However, studies have not shown whether estrogen can reduce uterine innervation and, if so, whether the time course is compatible with the rapid changes observed in the estrous cycle. These questions were addressed in the present study. Mature ovariectomized virgin rats received 17-beta-estradiol as a single injection (10 microg/kg s.c.) or chronically from timed-release pellets (0.1 microg/pellet for 3 weeks sustained release). Total (protein gene-product 9.5-immunoreactive) and sympathetic (dopamine beta-hydroxylase-immunoreactive) uterine innervation was assessed quantitatively. Both total and sympathetic innervation was abundant in uterine longitudinal smooth muscle of ovariectomized rats. However, following acute or chronic estrogen administration, total and sympathetic fiber numbers were markedly decreased. This was not due to altered uterine size, as reductions persisted after correcting for size differences. Our results indicate that sympathetic nerves are lost from uterine smooth muscle after estradiol treatment in a manner similar to that seen in the intact animal during estrus and pregnancy. This suggests that the rise in estradiol prior to estrus is sufficient to deplete uterine sympathetic innervation.     

Association between expression of the H histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus

RHDV (rabbit hemorrhagic disease virus) is a highly virulentcalicivirus that has become a major cause of mortality in wildrabbit populations (Oryctolagus cuniculus). It binds to thehisto-blood group antigen (HBGA) H type 2 which requires an1,2fucosyltransferase for its synthesis. In rabbit, three 1,2fucosyltransferasesgenes are known, Fut1, Fut2, and Sec1. Nonfunctional allelesat any of these loci could potentially confer resistance toRHDV, similar to human FUT2 alleles that determine the nonsecretorphenotype and resistance to infection by various NoV strains.In this study, we looked for the presence of H type 2 on buccalepithelial cells of wild rabbits from two geographic areas underRHDV pressure and from one RHDV-free area. Some animals withdiminished H type 2 expression were found in the three populations(nonsecretor-like phenotype). Their frequency markedly increasedaccording to the RHDV impact, suggesting that outbreaks selectedsurvivors with low expression of the virus ligand. Polymorphismsof the Fut1, Fut2, and Sec1 coding regions were determined amonganimals that either died or survived outbreaks. The Fut2 andSec1 genes presented a high polymorphism and the frequency ofone Sec1 allele was significantly elevated, over 6-fold, amongsurvivors. Sec1 enzyme variants showed either moderate, low,or undetectable catalytic activity, whereas all variant Fut2enzymes showed strong catalytic activity. This functional analysisof the enzymes encoded by each Fut2 and Sec1 allele suggeststhat the association between one Sec1 allele and survival mightbe explained by a deficit of 1,2fucosyltransferase expressionrather than by impaired catalytic activity.     

Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnant and nonpregnant mice.

Cytokine secretion by endometrial cells from estrous and mated mice was measured using specific bioassays. The granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) contents of uterine intraluminal fluid were elevated greater than 20-fold and 250-fold respectively following mating, and both cytokines were synthesized in abundance in vitro by uterine cells harvested at estrus and on Day 1 of pregnancy. Synthesis was not impaired in genetically lymphocyte-deficient nude, SCID, or beige mice. To determine the cellular origin of the cytokines, a panning technique employing monoclonal antibodies against a range of leukocyte and other lineage markers was used to isolate uterine cell subsets in vitro. These experiments identified glandular and/or luminal epithelial cells as the major source of GM-CSF and IL-6 in estrous and pregnant uteri. Stromal fibroblasts also synthesized IL-6, as did macrophages in mated mice. Epithelial cells harvested from midgestation uteri secreted GM-CSF and IL-6 in quantities similar to those of cells from estrous and mated mice. Bioactivities of both cytokines derived from epithelial cells were neutralized by specific antibodies, and size-exclusion chromatography of conditioned media from uterine cells revealed peaks of GM-CSF and IL-6 bioactivity with M(r) 23,000 and 23,000-26,000, respectively. Bioassay of luminal fluids and culture supernatants were negative for the cytokines interleukin-1, interleukin-2, interleukin-3, and tumor necrosis factor-alpha. These studies identify murine uterine epithelium as a potent source of the cytokines GM-CSF and IL-6, which we postulate have potentially important functions in pregnancy through actions on target cells in both the uterus and the conceptus.     

Molecular cloning, genomic mapping, and expression of two secretor blood group alpha (1,2)fucosyltransferase genes differentially regulated in mouse uterine epithelium and gastrointestinal tract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal alpha(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three alpha(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1 demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an alpha(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2 mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2 expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fucalpha1-->2Galbeta epitopes proposed to mediate blastocyst adhesion.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.     

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

H-Type 1 carbohydrate antigen expression by ovine endometrial cells

Our objective was to determine whether H-Type 1 carbohydrate antigen is expressed by ovine endometrial epithelial cells. Endometrium was obtained from sheep on days (D) 1, 5, 11, 13, and 15 of the estrous cycle or pregnancy and D17 and D19 of pregnancy. Immunofluorescence microscopy of frozen tissue sections revealed intense staining on the apical surface of glandular uterine epithelial (GE) cells from D11 to D17 of pregnancy. Light punctate staining of luminal uterine epithelial (LE) cells was present from D15 to D19 of pregnancy, with isolated areas of intense staining observed only on D15 of pregnancy. There were no noticeable differences in staining patterns on equivalent d of the estrous cycle. Immortalized sheep LE and GE cells were used to determine whether estradiol (E), progesterone (P), or E + P, with or without interferon tau (IFNtau), regulates H-Type 1 antigen expression in vitro. Intermittent punctate surface staining was observed on both cell lines independent of steroid treatment. Treatment with P or IFNtau increased H-Type 1 antigen expression (P < 0.01) and resulted in large aggregates of punctate staining. Domain-specific biotinylation and Western blotting of cell lysates from LE and GE cells were used to identify proteins carrying the H-Type 1 antigen. For both cell types, major immunoreactive apical membrane proteins were detected at 31, 33, 42, 55, 60, and 70 kDa. Therefore, the H-type 1 antigen is expressed mainly on GE cells during pregnancy recognition in utero and up-regulated by P and IFNtau on LE and GE cells in vitro.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Estrous expression during a fixed-time artificial insemination protocol enhances development and interferon-tau messenger RNA expression in conceptuses from Bos indicus beef cows

Expression of estrus (EST) near the time of fixed-time artificial insemination (FTAI) increases pregnancy success in beef females. This outcome has been associated with improved pregnancy establishment and maintenance, although research is still warranted to validate this theory. Hence, this experiment compared ovarian, uterine and conceptus factors associated with pregnancy establishment in Bos indicus beef cows according to estrous expression during a FTAI protocol. One hundred lactating multiparous Nelore cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device on day −11, a 12.5 mg injection of prostaglandin F_2α on day −4, P4 device removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of equine chorionic gonadotropin on day −2, and FTAI on day 0. An estrous detection patch was attached to the tailhead of each cow on day −2, and estrous expression was defined as removal of >50% of the rub-off coating from the patch at FTAI. Overall, 39 cows expressed EST, 55 did not express EST (NOEST), and six cows lost their patch and were discarded from the experiment. Ovarian ultrasonography was performed at FTAI, and on days 7 and 15 of the experiment. Blood samples were also collected on days 7 and 15. Only cows without a corpus luteum (CL) on day 0, and with a CL on days 7 and 15 remained in the experiment (EST, n=36; NOEST, n=48). On day 15, cows were randomly selected within each group (EST, n=29; NOEST, n=30) for conceptus collection via transcervical flushing, followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Within cows not assigned to conceptus collection, blood samples were collected for whole blood RNA extraction (day 20) and pregnancy status was verified by transrectal ultrasonography (day 30). Diameter of dominant follicle on day 0 and plasma P4 concentrations on day 7 were greater (P⩽0.02) in EST v. NOEST cows. Conceptus length and messenger RNA (mRNA) expression of prostaglandin E synthase and interferon-tau were greater (P⩽0.04) in EST v. NOEST cows. Moreover, EST cows diagnosed as pregnant on day 30 had greater (P<0.01) blood mRNA expression of myxovirus resistance 2 on day 20 compared with NOEST. In summary, estrous expression near the time of FTAI enhanced pregnancy establishment factors in B. indicus cows, including conceptus development and mRNA expression of interferon-tau.