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Fibroblast growth factor-2 (FGF-2) induces gastrulation of rabbit blastocysts in vitro and is present in the uterine secretion at day 6 after mating. The following study was made in order to show if changes in the uterine FGF-2 concentration or in the FGF receptor concentration of the embryonic tissues point to a regulation of this event. By the use of the ELISA technique and immunohistochemistry, FGF-2 concentration was determined in the endometrial tissue, uterine secretion and blastocyst between day 4 and day 8 of pregnancy, in the uterine secretion after induction of pseudopregnancy, in day 6 blastocysts after in vitro culture, and FGF immunoreactivity was localized in the endometrial tissue. FGF receptor-1 (FGFR-1) concentration was examined correspondingly in the blastocyst. Cross-linking experiments using 125I-FGF-2 were done to identify binding proteins in the blastocyst. In the uterine secretion, FGF-2 was constantly high up to day 6.5 but showed an increase thereafter. Similar values in pseudopregnant uterine secretions indicated that the growth factor was of uterine origin. It was probably synthesized by the uterine epithelium as shown by immunohistochemistry. Under culturing conditions, the blastocyst produced small amounts of FGF-2. In the blastocyst, FGFR-1 as well as binding of 125I-FGF-2 showed a dramatic increase from day 6.0 to day 6.5, coinciding with the onset of gastrulation. Receptor antigenicity was located in the embryonic disc at day 6.5 and day 7.0. Two binding proteins of about 200 and 130 kDa were found by cross-linking. The results indicate that a regulation of growth factor influence on embryonic differentiation is more probable via expression of the embryonic receptor than via differential release of the uterine growth factor.

The localization and release of uteroglobin (UGL) were investigated immunohistologically in the oviducts and uteri of female rabbits from oestrus through the 7th day post coitum and the blastocyst on the 7th day post coitum. UGL was detected within Fallopian tube cells even during oestrus. Granules of UGL appeared toward the bases of these cells. Subsequently, the cells became almost entirely filled with UGI. Drop-like protrusions of the apical cytoplasm suggest a mechanism of apocrine extrusion. All stages of filling and extrusion were visible during the entire preimplantation period. During oestrus, synthesis of UGL within uterine cells becomes sufficiently advanced so that extrusion has either already begun or is about to begin. UGL positive material first appears in the supranuclear regions. Later the entire cytoplasm shows a positive reaction. An uneven distribution of UGL cells is observed in the endometrium. Since only the glands adjacent to the myometrium and cells of the cavum epithelium contain UGL, a striking mosaic of UGL positive and negative cells results. The present report is the first detecting UGL in single cells of the blastocyst. Both entodermal and ectodermal cells proved to be UGL positive. The synthesis and section of UGL in the oviduct and uterus and the possible origins of UGL in the blastocyst are discussed.

beta-Glycoprotein was isolated from preimplantation uterine secretions of the rabbit by gel- and ion-exchange chromatography. Two fractions, called DF1 and DF2, were analyzed by isoelectric focusing (IEF) and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with Western blotting and immunoelectrophoresis. DF1 displayed 21 bands with isoelectric points of pH 5.2-7.6, and DF2 15 bands of pH 4.2-5.7. SDS-PAGE yielded up to 14 bands with major components at molecular weights of 63,000 and 135,000 respectively. Two-dimensional gel electrophoresis of DF2 in combination with Western blotting revealed five groups of proteins of equal molecular weights but with different isoelectric points, indicating immunological identities. Glycosidase activities in uterine secretions before and after implantation were studied and compared with those of the blastocyst fluids. alpha-L-Fucosidase co-eluted with DF1, and beta-N-acetylglycosaminidase was distributed in DF1 and DF2. Both enzymes were localized on isoelectric focusing gels, and N-acetylglucosaminidase was also demonstrated in an immunoprecipitate of DF1.

Two antigens of the beta-glycoprotein fraction from rabbit uterine secretion of the seventh day post coitum were purified firstly by gel filtration on Sephadex G 150, then either by ion exchange chromatography on DEAE Sephacel or chromatofocusing on PBE 94. By the use of a specific antiserum, raised in female sheep, two antigens with alpha 2- and beta 2-mobility in agar gel electrophoresis could be demonstrated. Immunohistochemical staining of the uterine epithelium at the seventh and eighth day post coitum showed the antigens to be localized in a ciliated cell type of conspicuous shape, which is supposed to be the site of synthesis. Stain accumulated mainly in the apical part of the cell, but there were also small deposits around the nucleus.

[3H] progesterone was injected into the uterine lumen of rabbits toward the end of preimplantation period (162 h post coitum). Light-microscopic autoradiography showed accumulation of label in single cell groups of the uterine epithelium. Fluorographs of thin layer chromatograms of steroid extracts indicated the metabolization of progesterone in the uterine tissue. Incubation of uterine sections with fluorescein isothiocyanate-conjugated progesterone-rabbit serum albumin revealed binding sites for this reagent: 162 h post coitum, staining was also localized in single cell groups of the uterine epithelium. Pretreatment with a monospecific antiserum showed uteroglobin to be the binding protein.

The synthesis of glycoproteins in rabbit uterine epithelium during the late preimplantation period was studied using tritiated N-acetylglucosamine. In vivo labelling was achieved by the intra-uterine implantation of agar gel columns containing the precursor. Autoradiography showed the radioactivity to be predominantly localized in the apical cell surfaces, with single cells exhibiting an accumulation of silver grains in their supranuclear cytoplasm. After gel electrophoresis of uterine flushings, activity was mainly found in the beta-glycoprotein fraction. Fluorescein isothiocyanate (FITC)-conjugated wheat-germ agglutinin reacted with the apical cytoplasm and surfaces of the endometrial cells. However, FITC-conjugated concanavalin A exhibited a different binding pattern, reacting first with the basal cytoplasm, and later with the apical cytoplasm. After concanavalin-A staining, single cells exhibited positive vesicles in their lateral and apical parts. These cells may be released into the uterine lumen until 210 h post column. Neither of the lectins reacted with ciliated cells. Concanavalin A showed an affinity for the beta-glycoprotein fraction of the uterine secretion. The results indicate that, although all endometrial cells contain glycoproteins, only a few of these seem to be involved in the synthesis of secretory products.

Rabbit uterine uteroglobin (UGL) was studied by electrophoretic and immunological methods following normal copulation, after ovariectomy and progesterone treatment, 17beta-oestradiol and combined progesterone treatment, 17 beta-oestradiol treatment alone and after HCG-induced pseudopregnancy. Electrophoretic studies show the amount of ULG in uterine secretions, the immunological investigations indicate the intracellular localization of ULG and the distribution of ULG-positive cells in the endometrium. No obvious differences were found between the uteri 7 days after injection with chorion-gonadotropin and those 7 days following normal copulation. No differences could be domonstrated between the uteri of animals 35 days following ovariectomy and subsequent progesterone treatment on Days 31-33 and those of normal 7 d. post coitum (p.c.) animals. Uteri from animals treated with progesterone on Days 2-5 p.c. contained more ULG-positive cells than controls. 17beta-oestradiol treatment with and without subsequent progesterone treatment resulted, in both gravid and ovariectomized animals, in the formation of a tall columnar endometrial epithelium. Treatment with 17 beta-oestradiol on Days 1 and 2 p.c. led to a decrease in the number of UGL-positive cells at 7 days p.c. Even after ovariectomy with 17 beta-oestradiol substitution, UGL-positive cells were still present in the endometrium. However a secretion of any magnitude could not be detected. The importance of differentiation between synthesis and secretion (= release) as distinct phases of the glandular response is especially emphasised by the latter findings.