Gas1 Regulates Patterning of the Murine and Human Dentitions through Sonic Hedgehog.

The mammalian dentition is a serially homogeneous structure that exhibits wide numerical and morphological variation among multiple different species. Patterning of the dentition is achieved through complex reiterative molecular signaling interactions that occur throughout the process of odontogenesis. The secreted signaling molecule Sonic hedgehog (Shh) plays a key role in this process, and the Shh coreceptor growth arrest-specific 1 (Gas1) is expressed in odontogenic mesenchyme and epithelium during multiple stages of tooth development. We show that mice engineered with Gas1 loss-of-function mutation have variation in number, morphology, and size of teeth within their molar dentition. Specifically, supernumerary teeth with variable morphology are present mesial to the first molar with high penetrance, while molar teeth are characterized by the presence of both additional and absent cusps, combined with reduced dimensions and exacerbated by the presence of a supernumerary tooth. We demonstrate that the supernumerary tooth in Gas1 mutant mice arises through proliferation and survival of vestigial tooth germs and that Gas1 function in cranial neural crest cells is essential for the regulation of tooth number, acting to restrict Wnt and downstream FGF signaling in odontogenic epithelium through facilitation of Shh signal transduction. Moreover, regulation of tooth number is independent of the additional Hedgehog coreceptors Cdon and Boc, which are also expressed in multiple regions of the developing tooth germ. Interestingly, further reduction of Hedgehog pathway activity in Shhtm6Amc hypomorphic mice leads to fusion of the molar field and reduced prevalence of supernumerary teeth in a Gas1 mutant background. Finally, we demonstrate defective coronal morphology and reduced coronal dimensions in the molar dentition of human subjects identified with pathogenic mutations in GAS1 and SHH/GAS1, suggesting that regulation of Hedgehog signaling through GAS1 is also essential for normal patterning of the human dentition.

WNT10A mutation results in severe tooth agenesis in a family of three sisters.

OBJECTIVES: To identify the genetic basis of severe tooth agenesis in a family of three affected sisters. PATIENTS AND METHODS: A family of three sisters with severe tooth agenesis was recruited for whole-exome sequencing to identify potential genetic variation responsible for this penetrant phenotype. The unaffected father was tested for specific mutations using Sanger sequencing. Gene discovery was supplemented with in situ hybridization to localize gene expression during human tooth development. RESULTS: We report a nonsense heterozygous mutation in exon 2 of WNT10A c.321C>A[p.Cys107*] likely to be responsible for the severe tooth agenesis identified in this family through the creation of a premature stop codon, resulting in truncation of the amino acid sequence and therefore loss of protein function. In situ hybridization showed expression of WNT10A in odontogenic epithelium during the early and late stages of human primary tooth development. CONCLUSIONS: WNT10A has previously been associated with both syndromic and non-syndromic forms of tooth agenesis, and this report further expands our knowledge of genetic variation underlying non-syndromic forms of this condition. We also demonstrate expression of WNT10A in the epithelial compartment of human tooth germs during development.

Diagnostic Accuracy of Symptoms, Endoscopy, and Imaging Signs of Chronic Rhinosinusitis Without Nasal Polyps Compared to Allergic Rhinitis.

Objectives The diagnosis of chronic rhinosinusitis without nasal polyps (CRSsNP) and distinguishing it from allergic rhinitis is difficult. Yet, early detection of CRSsNP is important to prevent progressive and severe chronic rhinosinusitis. Our aim was to compare diagnostic accuracy of symptoms, endoscopy, and imaging signs of CRSsNP and allergic rhinitis -only phenotypes. Setting Prospective controlled follow-up study. Participants Forty-two nonsmoking patients visiting tertiary care due to CRSsNP and 19 nonsmoking volunteer controls with allergic rhinitis filled a symptoms questionnaire and underwent nasal endoscopy off-seasonally. All CRSsNP patients underwent computed tomography scans of paranasal sinuses. All the allergic rhinitis control subjects and 14 of the CRSsNP patients underwent sinus magnetic resonance imaging. Results Radiologic Lund-Mackay score, duration of symptoms, visual analogue scale scores of symptoms, and Sinonasal Outcome Test 22 were significantly higher in the CRSsNP group compared to allergic rhinitis control group. These factors also correlated in part with each other. Endoscopic score did not correlate with other factors, nor did it differ between CRSsNP and allergic rhinitis groups. The highest area under curve value was demonstrated for visual analogue scale score of facial pain/pressure (0.93) and score ≥4/10 showed 60% sensitivity and 95% specificity for detecting CRSsNP group ( P < .001). Radiologic sign of obstructed osteomeatal complex showed 100% specificity and 38% sensitivity for detecting CRSsNP group ( P < .001). Conclusions CRSsNP phenotype could be primarily distinguished from allergic rhinitis by higher facial pain/pressure score and secondarily by radiologic sings of obstructed ostiomeatal complex and higher Lund-Mackay score. Endoscopic score has limited value in distinguishing CRSsNP from allergic rhinitis.

Vax1 Plays an Indirect Role in the Etiology of Murine Cleft Palate.

Cleft lip with or without palate (CLP) and isolated cleft palate (CP) are common human developmental malformations with a complex etiology that reflects a failure of normal facial development. VAX1 encodes a homeobox-containing transcription factor identified as a candidate gene for CLP in human populations, with targeted deletion in mice associated with multiple anomalies, including disruption of the visual apparatus and basal forebrain, lobar holoprosencephaly, and CP. We have investigated Vax1 function during murine palatogenesis but found no evidence for a direct role in this process. Vax1 is not expressed in the developing palate and mutant palatal shelves elevate above the tongue, demonstrating morphology and proliferation indices indistinguishable from wild type. However, mutant mice did have a large midline cavity originating from the embryonic forebrain situated beneath the floor of the hypothalamus and extending through the nasal cavity to expand this region and prevent approximation of the palatal shelves. Interestingly, despite strong expression of Vax1 in ectoderm of the medial nasal processes, the upper lip remained intact in mutant mice. We found further evidence of disrupted craniofacial morphology in Vax1 mutants, including truncation of the midface associated with reduced cell proliferation in forebrain neuroectoderm and frontonasal mesenchyme. Sonic hedgehog (Shh) signal transduction was downregulated in the mutant forebrain, consistent with a role for Vax1 in mediating transduction of this pathway. However, Shh was also reduced in this region, suggestive of a Shh-Vax1 feedback loop during early development of the forebrain and a likely mechanism for the underlying lobar holoprosencephaly. Despite significant associations between VAX1 and human forms of CLP, we find no evidence of a direct role for this transcription factor in development of this region in a mutant mouse model.

Low lymphatic vessel density associates with chronic rhinosinusitis with nasal polyps.

OBJECTIVES: Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP) and antrochoanal polyps (ACP) are different upper airway inflammation phenotypes with different pathomechanisms. In order to understand the development of tissue edema, the present study aimed to evaluate lymphatic vessel density in CRSsNP, CRSwNP and ACP. MATERIALS AND METHODS: 120 retrospective nasal and maxillary sinus specimens were stained immunohistochemically with a von Willebrand factor polyclonal antibody recognizing vascular and lymphatic endothelium, and with a podoplanin monoclonal antibody recognizing lymphatic endothelium. Vessels were studied by microscopy in a blinded fashion, and the vessel density and the relative density of lymphatic vessels were calculated. Patient characteristic factors and follow-up data of in average 9 years were collected from patient records. RESULTS AND CONCLUSION: In the nasal cavity, the low absolute and relative density of vessels and of lymphatic vessels was associated with CRSwNP and ACP tissues compared to control inferior turbinate. This was observed also in the inflammatory hotspot area. In the maxillary sinus, lower absolute and relative density of lymphatic vessels associated with the CRSwNP phenotype. High lymphatic vessel density in polyp tissue associated with the need for revision CRS-surgery. As a conclusion, low density of lymphatic vessels distinguished patients with CRSwNP not only in the hotspot area of polyp tissue, but also in maxillary sinus mucosa. Yet, higher lymphatic vessel density seems to associate with polyp recurrence. Further studies are still needed to explore if formation of nasal polyps could be diminished by intranasal therapeutics affecting lymphangiogenesis.

Glycodelin-A modulates syncytialization of human BeWo choriocarcinoma cell line.

Cytotrophoblasts are the key trophoblast cells which differentiate into different trophoblast lineages. In this report, glycodelin-A action on fusion of a cytotrophoblast-like cell line (BeWo) was investigated. It significantly reduced the spontaneous fusion of BeWo cells. The treatment enhanced the invasion and extracellular-signal regulated kinases activation of BeWo cells. The mRNA expression of syncytialization markers, human chorionic gonadotrophin and glial cells missing homolog 1 were suppressed upon glycodelin-A treatment. The data suggest a possible function of glycodelin-A in mediating cytotrophoblast differentiation.

Glycodelin in reproductive endocrinology and hormone-related cancer.

Glycodelin is an endocrine-regulated glycoprotein that has significant effects on immune cells, apoptosis, reproduction, cell adhesion, differentiation and cancer. In reproduction, glycodelin contributes to capacitation and immunoprotection of spermatozoa, and it modulates sperm-oocyte binding, acrosome reaction and implantation. In endocrine-related cancer, the differentiation inducing effects of glycodelin are accompanied by growth restriction of malignant cells, decreased expression of oncogenes, increased expression of tumour suppressor genes and morphological reversion of the malignant phenotype. This review features these properties and clinical connections, highlighting the role of glycosylation in biological actions.

Biogas from energy crops--optimal pre-treatments and storage, co-digestion and energy balance in boreal conditions.

The objective of this research was to evaluate the biogas production from crops in boreal conditions, focusing on the optimal pre-treatment and storage methods, co-digestion and energy balance of farm-scale crop based biogas plants. Alkaline treatments offered some potential for improving the methane yield from grass and sugar beet tops. The results show that the CH4 yield of energy crops can be maintained by appropriate ensiling conditions for even after 11 months in ambient conditions. The CH4 yield was best preserved with wet grass mixture without additives. Co-digestion of manure and crops was shown to be feasible with feedstock volatile solids (VS) containing up to 40% of crops. The highest specific methane yields of 268, 229 and 213 l CH4 kg(-1) VSadded in co-digestion of cow manure with grass, sugar beet tops and straw, respectively, were obtained during feeding with 30% of crop in the feedstock, corresponding to 85-105% of the total methane potential in the substrates as determined by batch assays. The energy output:input ratio of farm-scale grass silage based biogas plant varied significantly (3.5-8.2) with different assumptions and system boundaries being lowest when using only inorganic fertilizers and highest when half of the heat demand of the system could be covered by metabolic heat.

Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations.

Glycodelin is an example of a glycoprotein whose complex-type glycans mediate biological actions in human reproduction and immune reactions. Being attached to an identical protein backbone, glycodelin oligosaccharides vary significantly from one reproductive tissue to another and have an effect on its own secretion and role in cell communication. For instance, uterine glycodelin-A inhibits sperm-oocyte interaction by binding on the sperm head. This is a glycosylation-dependent phenomenon, in which fucosyltransferase-5 plays a key role. Glycodelin-S from seminal plasma binds evenly around the sperm head and maintains an uncapacitated state in the spermatozoa, until the isoform is detached during sperm passage through the cervix. Glycodelin-F from follicular fluid and Fallopian tube binds to the acrosomal region of the sperm head, thereby inhibiting both the sperm-oocyte binding and premature progesterone-induced acrosome reaction. The cumulus cells surrounding the oocyte can capture glycodelin-A and -F from the surrounding environment and convert these isoforms to a cumulus cell isoform, glycodelin-C. It differs by glycosylation from the other isoforms, and it too attaches on the sperm head, with the highest density in the equatorial region. Glycodelin-C is capable of detaching the sperm-bound inhibitory isoforms so that the sperm-oocyte binding is enhanced. Glycodelin-A also has immunosuppressive actions directed to cellular, humoral and innate immunity. Although these actions depend mainly on the protein backbone, glycosylation also plays a part. Glycosylated glycodelin may be involved in the protection of spermatozoa against maternal immune reactions, and glycodelin also has apoptogenic activity. Some glycosylation patterns of glycodelin may mask its apoptogenic domain. This review updates the recent research and clinical associations of glycodelin, highlighting the role of glycosylation.

Advances in uterine protein research: reproduction and cancer.

Uterine protein research has advanced from the measurements of specific compounds to detailed analysis of the genes that regulate protein synthesis and build up the complex carbohydrate structures that play important functional roles. Some 80% of all human proteins are glycoproteins. Functional glycomics highlights the importance of glycosylation in glycoprotein function. Glycodelin is a representative example of functional glycomics because its various glycoforms have different functions. In the uterus, synthesis of glycodelin-A is temporally regulated by progesterone. During the estrogen-dominated fertile window, absence of glycodelin synthesis is significant because uterine glycodelin-A potently and dose-dependently inhibits sperm-egg binding, the initial step in fertilization. The anti-fertilization propensity of glycodelin-A during the luteal phase of the cycle is highly glycosylation-dependent, and there is an intricate functional interplay between spermatozoa, zona pellucida and the various glycodelin isoforms present in the uterine fluid, seminal plasma and follicular fluid, respectively. Endometrial glycodelin synthesis can be induced during the fertile window by administration of progestagens, such as in levonorgestrel hormone-releasing IUD and contraceptive implants. Glycodelin can be chemically modified in such a fashion that it blocks the binding site on CD4 for the HIV surface glycoprotein, synthesis of viral gp 120, and infection of peripheral blood mononuclear cells by the primary HIV isolate THA/93/051, thus potentially inhibiting HIV transmission. Now that a cell line producing the contraceptive isoform has been identified by recombinant technology, these findings may have application for locally applied antiviral contraception. Glycodelin also has immunosuppressive properties, suggesting that the recognition mechanisms in immune and reproductive systems may have converged. Given its inhibitory activity on natural killer cells, abundant at the fetomaternal interphase, the high glycodelin concentration at the same site suggests a role in fetomaternal defense mechanisms. This may be relevant in women with recurrent miscarriage, in whom both the serum and uterine fluid glycodelin concentrations are decreased. Experiments on cancer cell lines have demonstrated increased epithelial differentiation by glycodelin cDNA transfection, and also by co-culture of cancer cells with normal stromal cells in the presence of basement membrane components. Both approaches result in glycodelin expression concomitant with decreased cell proliferation and reversion of the malignant phenotype. These results suggest an active role of normal stromal cells, basement membrane components and glycodelin in epithelial differentiation and glandular morphogenesis. This disposition of glycodelin is significant in patients with certain carcinomas, in which glycodelin-expressing tumors carry better prognosis than glycodelin-negative tumors of the same clinical stage and histological grade. Research on functional glycomics continues to produce significant information on fundamental aspects of fertilization, implantation, pregnancy and cancer.

The contribution of D-mannose, L-fucose, N-acetylglucosamine, and selectin residues on the binding of glycodelin isoforms to human spermatozoa.

Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.

Zona-binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin.

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.

High serum insulin-like growth factor binding protein-1 is associated with increased cardiovascular mortality in elderly men.

Insulin-like growth factor binding protein-1 (IGFBP-1) has been implicated in the development of cardiovascular disease, but it is not known whether IGFBP-1 is related to cardiovascular mortality. We examined the relation of circulating IGFBP-1 to death from coronary heart disease, cardiovascular disease, and all causes in a cohort study consisting of 622 men aged 65 - 84 years, at baseline in 1984. Fasting serum IGFBP-1 and other risk factors were measured in 1984 and 1989. Cardiovascular events for those who died between 1984 and 1995 were analyzed, and cardiovascular diagnoses were coded centrally according to standardized procedures. Of the 622 men, 358 died between 1984 and 1995; 160 deaths were due to cardiovascular causes, 113 of which were coronary deaths. High fasting serum IGFBP-1 concentration (> 75 percentile) in 1984 was associated with increased five-year total mortality (OR 2.05, 95 % CI 1.41 - 2.99; p < 0.0002), cardiovascular mortality (OR 2.20, 95 % CI 1.37 - 3.50; p < 0.0009) and coronary heart disease mortality (OR 2.29, 95 % CI 1.35 - 3.88; p < 0.002). After adjustment for age, high serum IGFBP-1 concentrations still carried an increased risk of total mortality due to (OR 1.73, 95 % CI 1.16 - 2.59; p < 0.007), cardiovascular (OR 1.91 95 % CI 1.18 - 3.09; p < 0.008) and coronary heart disease (OR 2.02. 95 % CI 1.18 - 3.47; p < 0.01). In conclusion, high fasting serum IGFBP-1 is related to increased five-year total and cardiovascular mortality in elderly men.

The synthesis and fate of glycodelin in human ovary during folliculogenesis.

The ontogeny of glycodelin in human ovarian follicles during folliculogenesis was studied. Glycodelin immunoreactivity began to be detected in the granulosa cells and thecal cells of late secondary follicles. Immunoreactivity was also found in both the luteinized granulosa cells and cumulus cells obtained from women undergoing the assisted reproduction treatment. However, only the luteinized granulosa cells, and not the cumulus cells, expressed glycodelin mRNA. Results also showed that the cumulus cells took up radiolabelled glycodelin and partially deglycosylated some of it. Glycodelin (and a partially deglycolsylated form of glycoldelin) appeared to complex with two cytoplasmic or membrane components of the cumulus cells. The data also demonstrated that ZIF-1, a glycoprotein isolated from human follicular fluid, was immunologically similar to glycodelin. In conclusion, we suggest that glycodelin is synthesized in the granulosa cells of ovarian follicles at late secondary follicle stage. It then may be released into the follicular fluid from where it is taken up and partially modified by the cumulus cells.

Endometrial expression of glycodelin in women with levonorgestrel-releasing subdermal implants.

OBJECTIVE: To determine whether subdermal levonorgestrel implants induce endometrial expression of glycodelin. DESIGN: Cross-sectional, blinded study. SETTING: University clinic. PATIENT(S): One hundred and eight women with subdermal implants and 19 postmenopausal women. INTERVENTION(S): Endometrial biopsies, curettages, and hysterectomies. MAIN OUTCOME MEASURE(S): Endometrial glycodelin expression was examined through immunohistochemistry, in situ hybridization, and morphologic endometrial dating. RESULT(S): Overall, 80% of the endometrial specimens obtained from women with subdermal levonorgestrel implants stained positive for glycodelin. Endometrial morphology of these women showed proliferative (71%), inactive/weakly proliferative (19%), menstrual or regenerating (6.5%), and other patterns (2.8%). Of these, 79%, 71%, 100%, and 100% were glycodelin positive, respectively. Nineteen specimens were obtained during the midcycle when glycodelin is not normally expressed: of these, 89% stained positive for glycodelin. Implant-related amenorrhea was associated with endometrial glycodelin expression in 58% of the women, whereas the endometrium specimens obtained from women with postmenopausal hypoestrogenic amenorrhea contained no detectable glycodelin. CONCLUSION(S): Subdermal levonorgestrel implant use is often associated with endometrial expression of glycodelin. Because glycodelin has been shown to inhibit sperm-egg binding, the induction of glycodelin may contribute to the contraceptive activity of the implant.

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.

Endometrial markers of uterine receptivity utilizing the donor oocyte model.

BACKGROUND: Ethical constraints limit the ability to study peri-implantation phase human endometrium. In this study, the donor oocyte model was used to study candidate endometrial markers of uterine receptivity. METHODS: Archived, paraffin-embedded tissue obtained by endometrial biopsy during cycle days 21-23 of patients undergoing 'mock' hormonal treatment cycles were evaluated by standard histological criteria and immunohistochemical staining for alpha v beta 3 integrin and glycodelin. All of these patients (n = 101) had undergone a donor oocyte embryo transfer cycle utilizing the exact same hormonal protocol. RESULTS: Histological evaluation revealed 62 (61.3%) in-phase, 34 (33.7%) dyssynchronous, 2 (2.0%) immature and 3 (3.0%) advanced endometria. The clinical outcomes of patients with either in-phase or dyssynchronous endometria were similar. Very strong correlations were noted between endometrial glandular dating and either alpha v beta 3 integrin or glycodelin immunostaining intensity (P < 0.001 for both). Glycodelin and alpha v beta 3 integrin immunostaining intensities were also highly correlated with each other (P < 0.001). CONCLUSIONS: Throughout the time period corresponding to the putative window of maximal endometrial receptivity (cycle days 21-23) a dynamic process was observed in exogenous hormonal replacement cycles characterized by a rapid histological advancement of endometrial glandular elements as well as progressive alpha v beta 3 integrin and glycodelin expression.