Dora C. Miceli

An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes

A trypsin-like oviducal proteinase acting upon the vitelline envelope of Bufo arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavlin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.

EFFECT OF OVIDUCAL PROTEINASE UPON BUFO ARENARUM VITELLINE ENVELOPE. A FLUORESCENCE APPROACH

The intrinsic fluorescence of vitelline envelope from amphibian eggs was enhanced after having been treated with pars recta secretion fluid. At the same time the total number of binding sites for 8‐anilino‐1‐naphthalene sulfonate (ANS) was increased, with the affinity constant remaining unchanged. These observations are interpreted in terms of a structural change of the vitelline envelope submitted to pars recta fluid effect.

The vitelline envelope-to-fertilization envelope transformation in the toad Bufo arenarum.

Abstract An investigation of some changes associated with the transformation of the vitelline envelope into the fertilization envelope in the egg of the toad Bufo arenarum is reported here. In most of the experiments described, the parameter used to demonstrate these changes was the stability of structural integrity of isolated envelopes when submitted to different agents and conditions. The envelopes used for this purpose exhibited a high degree of purity and remained apparently unaltered by the isolation procedure. As a quantitative method to ascertain their solubility rate, the release of uv-absorbing materials into solution was determined. Compared to the vitelline envelope, the fertilization envelope has proven to be less soluble in water, more stable in the presence of the chaotropic ion thiocyanate, and less susceptible to digestion in the presence of sperm lysin, trypsin, and pronase. In Bufo arenarum , as in other species, the vitelline envelope appears to be composed of glycoproteins. In contrast to previous results, however, disulfide bonds do not seem to be involved in their structural integrity. Thus, experiments carried out with isolated envelopes as well as with envelopes in situ have demonstrated a lack of effect of disulfide bond breaking agents on envelope stability. Evidence is presented suggesting that the solubility of envelopes in mercaptan solutions, as reported by other laboratories, is likely to be the expression of artifactual results.

Hormonal regulation of an oviducal protein involved in Bufo arenarum fertilization

Abstract 1. 1. The most cephalic portion of a toads oviduct, called pars recta (PR) in Bufo arenarum, is involved in fertilization. A protein acting upon the vitelline envelope (VE) of coelomic oocytes has been identified in the PR secretion fluid and tissue of PR. This protein modifies the oocytes vitelline envelope, rendering it sensitive to sperm lysin and hence making coelomic oocytes fertilizable. 2. 2. The modulating influence of different estrogens, progesterone as well as testosterone on PR activity has been studied in ovariectomized toads. 3. 3. Following gonadectomy, the activity of PR declined at different rates according to the season in which ovariectomy was performed. 4. 4. Administration of estrogenic hormones resulted in complete reversal of the effect produced by castration. 5. 5. Among the estrogens tested, estradiol-17β proved to be the most potent stimulator of PR activity. The estradiol-induced increase in PR activity was dosage-dependent and detectable as early as 60 min after intralymphatic hormone injection. 6. 6. Actinomycin and cycloheximide prevented the estradiol-induced enhancement of PR activity. 7. 7. Whereas progesterone failed to exert any appreciable effect on PR activity, testosterone was capable of stimulating biological activity of PR in a dosage higher than that of estradiol. 8. 8. The serum concentration of estradiol-17β was determined in females captured in different months of the year, and the results lend some support to the assumption that the estradiol synchronizes the cycle of ovarian maturation and PR activity.

LEFTY2 expression and localization in rat oviduct during early pregnancy

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-β) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-β superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-β1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.

Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction.

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.

Teratoid tumors derived from mouse embryos grown in an immunologically privileged site

Mouse early embryos and embryo fragments were transplanted into an immunologically privileged site, consisting of a glass cylinder previously implanted under the skin of adult mice in order to test their tumor producing potential, in allogeneic adult recipients. The highest yield of tumors was obtained upon transplantation of 6 1/2 day old embryos in toto. i.e., including the embryonic and extraembryonic areas. Histological examination showed teratomas composed of differentiated tissues derived from the three germ layers containing isolated foci of undifferentiated cells and nodules of trophoblast giant cells. Areas exhibiting the histological appearance of yolk sac carcinoma were also observed. Transplantation of the whole 6 1/2 day old egg cylinder, including the ectoplacental cone, and the isolated embryonic area produced a lower incidence of teratomas with a reduced variety of differentiated tissues. No yolk sac carcinoma was found in these grafts. The ectoplacental cone of 6 1/2 day embryos produced no tumors. Grafts of genital ridges from 12 1/2 day embryos gave rise to teratomas with well differentiated tissues of embryonic and extraembryonic origin. Areas ressembling yolk sac carcinoma were also observed. The life span of trophoblastic giant cells within the glass cylinder was significantly longer than in other experimental systems.