KLK6 and KLK13 predict tumor recurrence in epithelial ovarian carcinoma.

BACKGROUND: The human kallikrein-related peptidase family consists of 15 genes. Twelve of these genes are overexpressed in ovarian cancer and may represent potential markers for diagnosis, prognosis, and/or response to treatment. The aim of this study was to determine the prognostic significance of kallikrein-related peptidase 6 (KLK6) and kallikrein-related peptidase 13 (KLK13) in epithelial ovarian cancer by quantifying gene expression levels with tumour pathology and patient survival data. METHODS: Total RNA was isolated from 106 patients diagnosed with primary ovarian cancer, as well as 8 normal ovary controls. Samples were analysed by quantitative real-time PCR for KLK6 and KLK13 expression. Correlation between kallikrein gene expression and clinical characteristics was evaluated with the chi(2)-test. Survival analysis was performed using Kaplan-Meier and Cox proportional hazards regression models. RESULTS: Expression levels of both KLK6 and KLK13 mRNA were significantly increased in invasive cancers relative to normal ovaries (P=0.002 and 0.039 respectively). High KLK6 and KLK13 expression was an indicator of poor prognosis, with patients having a shorter recurrence-free survival (P=0.002 and 0.027 respectively). High KLK6 expression was also significantly associated with lower overall survival (P=0.011). When subjected to multivariate analysis, patients with either high KLK6 or KLK13 were 3- and 2.2-fold, respectively, more likely to have a recurrence than patients with low kallikrein expression. CONCLUSION: These data show increased mRNA expression of KLK6 and KLK13 in ovarian cancer compared to normal ovarian tissues. High KLK6 or KLK13 expression in primary ovarian tumours can significantly predict prognosis in terms of recurrence-free survival and overall survival. In all, this study shows KLK6 and KLK13 as potential biomarkers and may be therapeutic targets for treatment of ovarian cancer.

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

Transforming growth factor beta receptor signaling and endocytosis are linked through a COOH terminal activation motif in the type I receptor.

Transforming growth factor beta (TGF-beta) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-beta receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-beta-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-beta receptor complex.

Diabetes reduces right atrial beta-adrenergic signaling but not agonist stimulation of heart rate in swine.

This study assessed the effects of streptozotocin diabetes in swine on the heart rate response to beta-adrenergic stimulation the adenylyl cyclase signal transduction pathway. Diabetic animals (n = 9) were hyperglycemic compared to the control group (n = 10) (12.6 +/- 1.0 vs. 3.53 +/- 0.29 mM). There were no significant differences between the diabetic and nondiabetic groups in the heart rate response to isoproterenol, however, there was a significant reduction (14%) in beta-adrenergic receptor density in the right atrium in the diabetic (61 +/- 3 fmol/mg protein) versus the nondiabetic group (71 +/- 3) (P < 0.05). The content of guanosine triphosphate binding regulatory proteins (Gs and Gi) in the right atrium was not affected by diabetes, nor was adenylyl cyclase activity under unstimulated conditions or with receptor-dependent stimulation with isoproterenol. On the other hand, adenylyl cyclase activity was 34% lower when directly stimulated with forskolin, and it was reduced by 23% when stimulated through Gs with Gpp(NH)p. In conclusion, beta-adrenergic stimulation of heart rate with isoproteronol and the receptor-dependent signal transduction pathway remained intact in the right atrium of diabetic swine despite reduced beta-adrenergic receptor density, G-protein content, and direct stimulation of adenylyl cyclase activity.

Mechanisms of transforming growth factor-beta receptor endocytosis and intracellular sorting differ between fibroblasts and epithelial cells.

Transforming growth factor-betas (TGF-beta) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-beta type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-beta receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization.

Transforming growth factor-beta (TGF-beta) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-beta type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-beta type I receptors, the role of FKBP12 in TGF-beta receptor action is presently unclear. Using a chimeric TGF-beta receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-beta signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-beta receptor endocytosis.

Heteromeric and homomeric transforming growth factor-beta receptors show distinct signaling and endocytic responses in epithelial cells.

Transforming growth factor-beta (TGF-beta) induces distinct responses dependent upon the cellular context. It is unclear whether the initial receptor interactions identified in one cell type will be operative in another. Utilizing a chimeric receptor strategy we have examined the signaling and endocytic activity of both heteromeric (type I/type II) and homomeric (type I/type I or type II/type II) TGF-betaR interactions in Mv1Lu epithelial cells. In agreement with that observed in mesenchymal cells, all TGF-betaR signaling in Mv1Lu cells required the formation of a heteromeric type I-type II receptor complex. However, the initial endocytic response to TGF-betaR oligomerization was distinctly regulated in the two cell types. While heteromeric TGF-beta receptors were internalized and down-regulated, homomeric TGF-betaR interactions showed diminished endocytic activity in Mv1Lu cells. This contrasts to that observed in mesenchymal cultures where ligand bound to TGF-betaR homomers was internalized, yet the receptors were not down-regulated. Moreover, while previous reports have suggested that mutations at serine 172 or threonine 176 in the type I TGF-betaR separated transcriptional from proliferative responses, we found no separation of pathways or effect on initial endocytic activity when the analogous mutations were made in the chimeric receptors.

Differential requirement for type I and type II transforming growth factor beta receptor kinase activity in ligand-mediated receptor endocytosis.

Transforming growth factor beta (TGFbeta) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGFbeta type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGFbeta receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor alpha and beta receptors spliced to the TGFbeta type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGFbeta receptor internalization, down-regulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGFbeta receptors. They did not, however, modulate TGFbeta signaling stimulated through the endogenous TGFbeta receptor. Although TGFbeta receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGFbeta receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.

Transforming growth factor beta and the endometrium.

During the oestrous or menstrual cycle and throughout much of pregnancy, the uterine endometrium undergoes rapid, as well as progressive, morphological and functional modification. During the preimplantation stage of pregnancy, the endometrium provides an environment that sustains embryonic development, and then participates in the nidation process. Later, the endometrium contributes the maternal component of the fetomaternal placenta. For a successful pregnancy, the placenta must orchestrate and regulate opposing forces. Trophoblast invasion must be limited to protect the uterus from destruction, while the allogenic fetus must be guarded from maternal immunological attack. Because of their powerful effects on the cellular and molecular processes associated with cellular proliferation and differentiation, angiogenesis and immunomodulation, the transforming growth factor beta (TGF-beta) polypeptides have been identified as potential modulators of many endometrial functions. Here, we examine the literature concerning cell-specific and temporal patterns of TGF-beta expression in the uterine endometrium during the oestrous cycle and pregnancy and evaluate the influence of ovarian steroids on TGF-beta expression in a range of species. Studies of the function of TGF-beta in the endometrium and at the fetomaternal interface are reviewed and discussed.

Immunolocalization of retinol-binding protein and profiles of its mRNA as related to testicular development in the beef bull.

A study was conducted to identify the cell types that express retinol-binding protein (RBP) in the bovine testis and to compare relative steady-state levels of RBP mRNA expression at different times of testicular development. At the ages of 10 (n = 3), 20 (n = 8), and 34 (n = 7) wk, Angus bulls were bled three times at 1.5-hr intervals, then surgically castrated. Blood samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), by radioimmunoassay and the degree of seminiferous tubule development was evaluated histologically in sections of fixed tissue samples stained with hematoxylin and eosin. Immunolocalization of RBP was based on the biotin-strepavidin-horseradish peroxidase method. Testis weight and concentrations of LH and T increased with age (P < 0.05), but those of FSH did not change (P > 0.05) between 10 and 34 wk. Seminiferous tubules at 10 wk contained immature Sertoli cells and gonocytes whereas, at 20 wk, spermatogonia and few spermatocytes were detected. At 34 wk, Sertoli cells appeared differentiated and spermatids were observed. RBP was immunolocalized in Sertoli, Leydig, and peritubular cells at the ages of 10, 20, and 34 wk. Furthermore, no differences in staining between Sertoli cells from tubules with or without germ cells were detected. Northern hybridization of testicular RNA with an RBP cDNA probe revealed the presence of a 1.4-Kb mRNA, which was similar to previous RBP transcripts found in other bovine tissues. Quantitative slot blot analysis revealed that steady-state RBP mRNA levels were 50% higher at 10 wk (P < 0.05) than at 20 and 34 wk of age.

Distinct endocytic responses of heteromeric and homomeric transforming growth factor beta receptors.

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

Developmental gene expression of procollagen III in bovine extraembryonic membranes during early pregnancy.

A major secretory protein produced by bovine chorioallantoic membranes, in vitro, was previously identified as the carboxyl-propeptide of alpha-1 type III collagen. In the present study, the protein and gene expression of procollagen III by bovine chorioallantois between days 17 and 45 of pregnancy was investigated. In addition, differential usage of multiple transcription termination sites by chorioallantois was examined. Two-dimensional PAGE of proteins synthesized and released by whole conceptuses or isolated chorioallantoic membranes into culture medium demonstrated that the C-terminal of procollagen III was not detectable before day 21 of pregnancy and concentrations increased thereafter. Developmental gene expression was determined by Northern blot analysis using a probe (A) that preceded all five polyadenylation sites of the previously sequenced clone 9.22. Procollagen III mRNA expression was undetectable at day 17, low on day 20, and increased through day 36. Two major transcripts of 5.9 and 4.9 kb were identified, the latter of which was expressed more prominently. A second probe (B), which terminated between poly-A sites 2 and 3, was designed to identify transcripts that terminated at poly-A site 1 or 2. This probe bound to the 5.9-kb mRNA only. Two additional procollagen III cDNA clones were isolated from our bovine conceptus cDNA library and sequenced. One, designated 9.29, terminated at poly-A site 5. The other, designated 11.7, terminated at poly-A site 2, indicating that the bovine conceptus uses these stop sites in procollagen III transcription. Results from this study demonstrate that procollagen III gene and protein expression coincide with the development of the allantois, which progressively fuses with the chorion forming the chorioallantois placenta. In addition, multiple termination sites are used in procollagen III transcription.

The role of trophoblast interferons in the maintenance of early pregnancy in ruminants.

PROBLEM: Are the effects of ruminant trophoblast interferon-tau (IFN-tau) on uterine prostaglandin (PG) secretion a specific action of this cytokine and what are the effects of IFN-tau on expression of uterine genes not generally associated with pregnancy maintenance? METHODS: The effects of IFN-tau and IFN-alpha on bovine uterine explant and epithelial cell production of PGF2 alpha and PGE2 were determined in the presence and absence of oxytocin (OT). The effects of intrauterine administration of IFN-tau were determined on uterine expression of retinol-binding protein (RBP) and transforming growth factor-beta (TGF-beta) isoforms. RESULTS: IFN-tau attenuated uterine endometrial secretion of PGF2 alpha and PGE2 in vitro and diminish PG stimulation by OT. IFN-tau and IFN-alpha were observed to be equipotent. Intrauterine infusion of IFN-tau resulted in a significant decrease in steady-state RBP mRNA levels and expression of TGF-beta 1, 2, and 3 mRNA levels were lowest in IFN-tau treated animals. CONCLUSION: Negative regulation of gene expression may be a general strategy in IFN activity. This may explain the similar activities of IFN-tau and IFN-alpha on a broad variety of cell types, including ruminant uterine endometrium.

Bovine endometrial retinol-binding protein secretion, messenger ribonucleic acid expression, and cellular localization during the estrous cycle and early pregnancy.

Retinol-binding protein (RBP) mRNA was localized to luminal and glandular epithelial cells of bovine endometrium by in situ hybridization. Relative levels of endometrial RBP mRNA expression during the estrous cycle and early pregnancy were determined by quantitative slot blot analysis and contrasted with uterine luminal concentrations of RBP. Expression of mRNA was moderate at Day 1 after estrus, declined at Day 5, reached lowest levels by Day 10, rose significantly through Days 15-17, and peaked at Day 20. RBP mRNA expression in pregnant animals was similar to that in cyclic animals on Day 15, doubled between Days 17 and 20, remained constant through Day 22, and rose slightly thereafter. Luminal RBP concentrations of cyclic cows, as determined by ELISA, decreased from Day 1 through Day 10, rose dramatically on Day 15, then declined through Day 20. Concentrations of RBP in uterine flushes from pregnant animals were similar to those of cyclic cows on Day 15 but remained relatively constant through Day 17. It is concluded that 1) RBP synthesis occurs in the luminal and glandular epithelial cells, 2) RBP transcription and secretion are correlated with each other, and 3) ovarian steroids, possibly in conjunction with uterine concentrations of their receptors, modulate uterine RBP expression.

Isolation and identification of porcine embryonic basic protein as a fragment of pregnancy-associated glycoprotein-2.

Between days 11 and 12 of gestation, the porcine conceptus undergoes a metamorphosis from a spherical blastocyst to an elongate thread-like form. During this process, the conceptus secretes a variety of products. One of these products is protein previously referred to as porcine embryonic basic protein (BP). This protein has been shown to be a major secreted product between days 13 and 18. In this study, we report a simple two-step procedure to isolate BP from day 15 porcine conceptus conditioned medium, utilized ion-exchange chromatography and reverse-phase HPLC. Purified BP was subjected to Edman degradation amino-terminal sequencing and a 25 amino acid residue sequence was obtained. Comparing the N-terminal sequence of BP to sequences in the GenBank database determined that BP shared amino acid homology with porcine pregnancy-associated glycoprotein-2 (PAG-2). The region of identity corresponded to an internal site of PAG-2, suggesting BP was a proteolytic fragment of PAG-2. The purified protein was confirmed to be BP by Western blot using a previously characterized anti-BP antiserum. Also, the BP was immunolocalized with the trophectoderm of day 11 blastocysts. Staining intensity was diminished in spherical blastocysts compared to elongated blastocysts. Although the function of PAG-2 and its cleavage product BP are unknown, the large quantity produced by the porcine conceptus and its sequence conservation across species may indicate a necessary role in early pregnancy.

Ovine endometrial expression of transforming growth factor beta isoforms during the peri-implantation period.

During the estrous cycle and early pregnancy, the uterus undergoes a variety o morphological changes. Because of their powerful effects on angiogenesis, on extracellular matrix modification, and on cellular proliferation and differentiation, the transforming growth factor beta (TGF beta) family of polypeptides may be involved in the regulation of pregnancy-related endometrial modification. In this study, endometrial stead-state mRNA expression of the three isoforms, TGF beta 1, beta 2, and beta 3, was quantified, and the proteins were localized during the later part of the estrous cycle (Day 13 and 16) and during early pregnancy (Days 13 through 30) in sheep. TGF beta 1 mRNA was expressed as a single transcript with steady-state mRNA expression levels 2-fold higher on Day 16 of the estrous cycle than on Day 13 of the estrous cycle (p < or = 0.002) or Days 13 and 16 (p < or = 0.004) and 0.008, respectively) of gestation. During pregnancy, levels increased progressively to peak on Day 27 (2.5-fold above Day 16 of pregnancy; p < or = 0.0001) and then leveled off at Day 30. Immunocytochemical localization of TGF beta 1 demonstrated protein in glandular protein in glandular and luminal epithelium at all days examined. TGF beta 2 mRNA was expressed as five distinct transcripts, and mRNA expression levels were lowest in Day 16 pregnant endometrium. Thereafter the expression level increased steadily through Day 30 (p < or = 0.0003). TGF beta 2 protein was localized in epithelium, diffusely within the endometrial stroma, and in leukocyte-like cells within the stroma. TGF beta 3 was expressed as one major transcripts and two minor transcripts. As with TGF beta 1, there was a dramatic difference in TGF beta 3 levels between Day 16 of pregnancy and Day 16 of the estrous cycle (3.8-fold, p < or = 0.0001). In pregnant ewes, endometrial TGF beta 3 levels increased 1.9-fold (p = 0.13) between Days 16 and 23 and remained relatively constant through Day 30. Immunohistochemistry localized TGF beta 3 protein most prominantly in the subepithelial stroma of the caruncule from Day 16 of the cycle in endometrial tissue. The observed changes in mRNA and protein expression patterns of TGF beta s within the ovine endometrium suggest that TGF beta s play a role in restructuring and modifying endometrium for a subsequent estrous cycle and/or pregnancy.

Early gestational expression of transforming growth factor beta isoforms by the ovine placenta.

The ruminant blastocyst metamorphoses from a 1-mm sphere to a 1 x 190-mm thread between Days 12 and 15 of gestation. The transforming growth factor beta (TGF beta) family of polypeptide growth factors are potential regulators of embryonic elongation because of their ability to regulate cellular replication, differentiation, and extracellular matrix formation. In mammalian development three isoforms of TGF beta-- TGF beta 1, beta 2, and beta 3--have been identified. Through the use of isoform-specific probes, ovine embryonic TGF beta 1 and beta 2 transcripts were identified and characterized in the present study. TGF beta 1 was expressed as a single transcript 2.6 kb in length. Levels increased by 14.8-fold from Day 13, when levels were barely detectable, to Day 27, when they were highest (p = 0.0001). Enriched samples of Day 20 chorionic and allantoic membranes demonstrated that the allantoic membrane contained 3.9-fold greater levels (p = 0.001) of TGF beta 1 message than the chorion. TGF beta 2 was expressed as five transcripts 6.2, 5.2, 4.8, 4.0, and 2.7 kb in length. From Day 13 to Day 23, there was a 6.2-fold increase (p = 0.0005) in TGF beta 2 expression; thereafter, expression declined 20% by Day 30 (p = 0.03). Extra-embryonic membrane expression of TGF beta 2 was slightly greater (1.5-fold; p = 0.01) in allantois than in chorion. Expression of TGF beta 3 was not detectable by Northern blotting, and only trace quantities of TGF beta 3 transcript were detected by slot-blot analysis. Antisera specific to TGF beta 1, beta 2 and beta 3 were used to immunolocalize the proteins within tissues. TGF beta 1 and beta 2 were identified in Day 16 trophectoderm and yolk sac. Both were also localized in chorion, allantois, and placental endothelium through Day 30. TGF beta 3 protein could not be detected in any conceptus tissue at any of the stages examined. The increase in expression of TGF beta 1 and beta 2 mRNA coincidental with conceptus elongation and placental development, as well as tissue localization of the protein, suggests their involvement in early gestational development in sheep.

Early gestational expression of retinol-binding protein mRNA by the ovine conceptus and endometrium.

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol-binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two-fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue-specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression.

Expression and cellular localization of retinol-binding protein messenger ribonucleic acid in bovine blastocysts and extraembryonic membranes.

A cDNA clone encoding retinol-binding protein (RBP) was isolated from a bovine conceptus cDNA library by use of an antiserum specific for bovine conceptus RBP (bcRBP). The RBP cDNA clone, designated bcRBP-700, is 732 bp in length and codes for a protein whose predicted amino acid sequence is identical to that of bovine plasma RBP. The size of the RBP mRNA transcript in bovine chorioallantois was approximately 1.4 kb as determined by Northern blot analysis. Expression of the protein and its mRNA in expanding bovine conceptuses (Day 13) and extraembryonic membranes (Day 45) was determined by immunocytochemistry with anti-bcRBP serum and in situ hybridization with 35S-labeled bcRBP-700 cDNA. Strong immunostaining for RBP and hybridization signals for RBP mRNA were observed in trophectoderm of tubular but not spherical Day 13 blastocysts. RBP mRNA was localized in epithelial cells lining the chorion, allantois, and amnion at Day 45 of pregnancy. In addition, RBP mRNA was detected in cotyledons, the sites of chorionic attachment to the uterine endometrium and physiological exchange between the embryo and its mother. Expression of RBP in expanding conceptuses, developing extraembryonic membranes, and sites of fetal-maternal attachment suggests that the extraembryonic membranes regulate retinol transport and availability within the conceptus.

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.