Genetic and QTL analyses of seed dormancy and preharvest sprouting resistance in the wheat germplasm CN10955.

The inheritance and genetic linkage analysis for seed dormancy and preharvest sprouting (PHS) resistance were carried out in an F8 recombinant inbred lines (RILs) derived from the cross between "CN19055" (white-grained, PHS-resistant) with locally adapted Australian cultivar "Annuello" (white-grained, PHS-susceptible). Seed dormancy was assessed as germination index (GI7) while assessment for preharvest sprouting resistance was based on whole head assay (sprouting index, SI) and visibly sprouted seeds (VI). Segregation analysis of the F2, F3 data from the glasshouse and the RIL population in 2004 and 2005 field data sets indicated that seed dormancy and PHS resistance in CN19055 is controlled by at least two genes. Heritabilities for GI7 and VI were high and moderate for SI. The most accurate method for assessing PHS resistance was achieved using VI and GI7 while SI exhibited large genotype by environment interaction. Two quantitative trait loci (QTLs) QPhs.dpivic.4A.1 and QPhs.dpivic.4A.2 were identified. On pooled data across four environments, the major QTL, QPhs.dpivic.4A.2, explained 45% of phenotypic variation for GI7, 43% for VI and 20% for SI, respectively. On the other hand, QPhs.dpivic.4A.1 which accounted for 31% of the phenotypic variation in GI7 in 2004 Horsham field trial, was not stable across environments. Physical mapping of two SSR markers, Xgwm937 and Xgwm894 linked to the major QTL for PHS resistance, using Chinese Spring deletions lines for chromosome 4AS and 4AL revealed that the markers were located in the deletion bins 4AL-12 and 4AL-13. The newly identified SSR markers (Xgwm937/Xgwm894) showed strong association with seed dormancy and PHS resistance in a range of wheat lines reputed to possess PHS resistance. The results suggest that Xgwm937/Xgwm894 could be used in marker-assisted selection (MAS) for incorporating preharvest sprouting resistance into elite wheat cultivars susceptible to PHS.

Expression of granulocyte-colony stimulating factor and its receptor is regulated during the development of the human placenta.

The development of the placenta is dependent upon the regulated proliferation, invasion and differentiation of trophoblast. Expression of cytokines at the feto-maternal interface suggests that these molecules may participate in placentation. The expression of granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) during the development of the human placenta was studied by immunohistochemistry using an anti-G-CSF monoclonal antibody (mAb) and two novel anti-G-CSFR mAbs. G-CSF was present in the stroma of fetal chorionic villi and maternal decidual tissues throughout pregnancy. G-CSFR was detected at high levels in fetal first and third, but not second trimester placental tissues. Staining for G-CSFR was undetectable in maternal decidual tissue from all gestational stages. In first trimester tissues, staining for placental G-CSFR was strongest in differentiated syncytiotrophoblast and invasive, extravillous cytotrophoblast, and weak staining was evident in undifferentiated cytotrophoblast. Immunohistochemical data suggesting temporal regulation of G-CSFR were corroborated by Western blotting and amplification by reverse transcription and PCR of G-CSFR mRNA. These data suggested that expression of G-CSFR in the human placenta is regulated both temporally and spatially, and that placental G-CSF is involved in paracrine regulation, and indicate a role for G-CSF and G-CSFR in trophoblast growth or function during placentation.

Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G.

Expression of MHC class I antigens on trophoblast populations in first trimester human chorionic villous tissue was assessed by immunohistology. Antibodies used were W6/32 which recognizes a non-polymorphic framework determinant of HLA- A, -B, -C, MHM5 specific for HLA-B, C and 4E and B23.1 which are specific for HLA-B. Syncytiotrophoblast and villous cytotrophoblast were negative with all the anti (HLA class I) antibodies tested. Interstitial trophoblast cells within the maternal decidua were identified with a new antibody, NDOG5, which is specific for extravillous cytotrophoblast. Double labelling showed that they bind W6/32 but not 4E, MHM5 or B23.1; consistent with the expression of the monomorphic HLA-G. In contrast the cytotrophoblast cells of the cell islands and cytotrophoblast shell, which also express the NDOG5 antigen, were positive with W6/32, 4E, MHM5 and B23.1. Cell column cytotrophoblast cells were negative with all four MHC class I antibodies. These results suggest that differentiation of cytotrophoblast from noninvasive to invasive forms is associated with transient expression of class I antigens other than HLA-G on cytotrophoblast shell and cell island cytotrophoblast.

Production of granulocyte colony-stimulating factor at the materno-foetal interface in human pregnancy.

A bioassay specific for human granulocyte colony-stimulating factor (G-CSF) was developed and used to measure G-CSF production in human pregnancy tissues. G-CSF was secreted by both foetal chorionic villous and maternal decidual tissues taken in the first trimester and at term. The level of G-CSF production by placental tissue was 6750 (1250-10,000) units of bioactivity per g of tissue in 48 hr in the first trimester and 104 (83-190) U/g at term. Bioactive G-CSF was also secreted by decidual tissue, more in the first trimester than at term. ELISA immunoassays measured 75 (10-820) ng/g/48 hr of G-CSF antigen from first trimester placenta, 15 (10-50) ng/g from first trimester decidua and less than 2 ng/g from term placenta. RNA isolated from decidual and chorionic villous tissue or from cells purified by flow cytometry, contained G-CSF mRNA in both tissues. In decidua, mRNA for G-CSF was confined to the macrophages, and cytotrophoblast from term amniochorion contained no detectable G-CSF mRNA. No G-CSF, measured as bioactivity or as mRNA, was detectable in choriocarcinoma cell lines.

Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres.

Isolation of pure preparations of the different cell populations of human endometrium is a prerequisite for studies of in-vitro function. Sieving of dispersed endometrial cells, followed by adsorption onto immunomagnetic microspheres coated with antibody to Thy-1 was used to separate glandular and stromal cells. The purity of these cell populations was checked with antibodies to cytokeratin and Thy-1. The stromal cells were 98% pure and 90% viable, gland cells were 82% pure with 76% viability. The purified cells were able to proliferate in vitro as shown by thymidine incorporation.

Purification of human cytotrophoblast from term amniochorion by flow cytometry.

Term cytotrophoblast do not express polymorphic MHC Class I antigens, unlike other fetal and maternal cells in the amniochorion/decidua. This allows cytotrophoblast to be isolated and purified from this tissue, utilizing 4E, a monoclonal antibody specific for HLA-B, which labels only non-trophoblast. We have developed a method using enzymic dispersion and Percoll gradient centrifugation, followed by flow cytometry, that yields, on average, a total of 5 X 10(6) term extravillous cytotrophoblast, 97 per cent pure. The availability of highly purified extravillous cytotrophoblast, for the first time, permits precise investigation of trophoblast function.