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Biomedical subjects

D R Armant

Publications and source records attributed to D R Armant.

At least 55 records · Page 3Linked to original sources
PubMed1990

Spectrophotometric analysis of follicular fluid related to oocyte fertilization, embryo cleavage, and follicular fluid protein and hormone content.

Spectrophotometric absorbance patterns of follicular fluids (FF) obtained from in vitro fertilization patients were analyzed and compared with oocyte fertilization and embryo cleavage rates. Each absorbance pattern was resolved into three components: delta OD360, delta OD415, and delta OD455. A positive correlation was found between fertilization outcome and delta OD455. The absorbances were not related to embryo cleavage rates. We confirmed the presence of bilirubin and beta-carotene in FF: these pigments absorb at 455 nm and are most likely serum-derived. To explain the association between delta OD455 and fertilization, we hypothesized that the FF delta OD455 is a marker of the degree of vascularization of the follicle that could be assessed by FF protein and hormone concentrations. However, no correlation was found between the delta OD455 and these FF parameters, suggesting an alternative explanation for this association.

Body Fluids↗
PubMed1989

Thin-layer chromatographic method for the determination of glycosyltransferase activity.

To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.

Animals↗
PubMed1989

Characterization of fucosyltransferase activity during mouse spermatogenesis: evidence for a cell surface fucosyltransferase.

Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltransferase activity of mixed germ cells displayed an apparent Vmax of 17 pmol (mg of protein)-1 min-1 and an apparent Km of approximately 13 microM for GDP-L-[14C]fucose in the presence of saturating amounts of asialofetuin at 33 degrees C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization.

Animals↗
PubMed1989

Absence of a direct effect of recombinant interleukins and cultured peritoneal macrophages on early embryonic development in the mouse.

On the basis of their recent studies, several researchers have suggested that the infertility associated with mild endometriosis is due to the alteration of peritoneal fluid, resulting in impairment of the viability of gametes or embryos. Elevated numbers of macrophages and lymphocytes have been reported in the peritoneal fluid of patients with endometriosis. Interleukin-1 (IL-1) a major product of activated macrophages and Interleukin-2 (IL-2), a product of most activated T-cells, have been postulated to play a role in the infertility associated with this disease, possibly by acting as direct embryotoxic agents. We have examined the effect of purified recombinant IL-1 and IL-2, which are not species-specific, on in vitro development of mouse embryos. Both interleukins had no effect on development to the blastocyst stage or on early stages of implantation, as measured in vitro by attachment and outgrowth of blastocysts to fibronectin-coated dishes. Moreover, co-culture of mouse embryos with activated human peritoneal macrophages had no effect on embryogenesis. We conclude that neither IL-1, nor other products of human macrophages activated by lipopolysaccharide, nor IL-2 are directly toxic to early mouse embryonic development.

Animals↗
PubMed1988

Spectrophotometric absorbance of follicular fluid: a predictor of oocyte fertilizing capability.

Follicular fluid volume, scoring of the oocyte-cumulus-corona-complex (OCCC), and spectrophotometric absorbance of the follicular fluid were separately compared between a group of fertilized (n = 53) and unfertilized oocytes (n = 35). Scoring of the OCCC and follicular fluid volumes was not found to be statistically different between the two groups. Spectrophotometric analysis of the follicular fluid in the visible spectrum demonstrated two peaks of maximum absorbance at 415 nm and 455 nm. The group of oocytes that fertilized was associated with follicular fluids that had significantly higher absorbances at 415 nm and 455 nm. In conclusion, follicular fluid volume and scoring of the OCCC were poor predictors of fertilizing capability; however, spectrophotometric absorbance, particularly at 455 nm, was positively correlated with oocyte fertilization.

Adult↗
PubMed1987

Quantitation of macromolecular binding using size exclusion filters: application to a fucosyltransferase assay.

A method to quantify the covalent attachment of small radiolabeled substrates to macromolecules on the basis of molecular weight using size exclusion filters in an Amicon Centrifree micropartition system is described. GDP-[14C]-L-fucose was covalently attached to asialofetuin in a fucosyltransferase reaction catalyzed by mouse spermatogenic cell extracts. Radiolabeled product was separated from unreacted substrate by centrifuging 200-400 microliter of cell extract through a 10-kDa size exclusion filter at 1000 g for 10 to 20 min. After 10 washes with an appropriate buffer, no detectable radioactivity was found in the eluant and the membrane-bound radiolabeled product was counted in a scintillation vial. Using this method the fucosyltransferase activity of mouse spermatogenic cells was approximately 17 pmol/mg protein/min which is essentially identical to values obtained using size exclusion chromatography. This technique provides a rapid, efficient, and inexpensive alternative for the isolation and detection of acceptor-substrate complexes.

Animals↗
PubMed1987

The impact of embryo quality and quantity on implantation and the establishment of viable pregnancies.

The role of cleavage rates, number of embryos transferred, and some other variables in pregnancy outcome in 222 human embryo transfers was studied. Pregnancy rates were significantly higher in a group of 117 patients receiving at least one embryo that had reached the four-cell stage at 40 hr postinsemination (26% total pregnancies/transfer and 18% ongoing pregnancies/transfer) than in the 105 patients receiving embryos developing at a slower rate (7% total pregnancies/transfer and 2% ongoing pregnancies/transfer). Of the 23 ongoing or term pregnancies produced, 21 came from transfers of at least one embryo that had reached four-cell stage by 40 hr postinsemination. Pregnancy rates were unaffected in either the fast-cleaving or the slower-cleaving embryos by culturing in vitro for an additional 24 hr. The presence of anucleate cell fragments also had no effect on pregnancy rates. Pregnancy rates increased progressively with the transfer of more embryos per transfer. These results suggest that procedures to improve ovulation induction and in vitro embryo culture technique may better the success of in vitro fertilization by providing a high number of rapidly cleaving embryos for transfer.

Cleavage Stage, Ovum↗
PubMed1987

The influence of exogenous human chorionic gonadotropin cycles with spontaneous luteinizing hormone surges on the outcome of in vitro fertilization.

Thirty consecutive patients who displayed endogenous luteinizing hormone (LH) surges and reached oocyte recovery for the purpose of in vitro fertilization were reviewed (LH surge group). Another 37 patients receiving human chorionic gonadotropin (hCG) in the absence of an LH surge served as a control group (no surge-hCG group). All patients underwent ovulation induction using clomiphene citrate (CC) plus human menopausal gonadotropin (hMG). The numbers of oocytes recovered and embryos transferred were significantly lower in the LH surge group compared with the no surge-hCG group (P less than 0.05). Nevertheless, the pregnancy rate per oocyte recovery between the two groups appeared similar (20 versus 16%). Among the 30 patients with a LH surge, 9 received hCG after a LH surge was verified, whereas the others did not. Addition of hCG to the LH surge increased the numbers of oocytes recovered, embryos transferred, and the pregnancy rate (3 out of 9 versus 3 out of 21); however, this difference is not statistically significant. Furthermore, the addition of exogenous hCG to a spontaneous LH surge appears to be necessary to maximize the number of oocytes recovered, embryos transferred, and pregnancies obtained.

Adult↗
PubMed1987

Fucosylation events during mammalian spermatogenesis.

It will be necessary to conduct further studies to establish more precisely the localization of FT on mouse male germ cells. Antibodies to FTs are not yet available, so an immunocytochemical approach is not currently feasible. Additional cell fractionation protocols can be designed to compare plasma membrane fractions with enriched fractions of Golgi apparatus and to compare directly the activities of multiple glycosyltransferase enzymes and Golgi-specific markers in these preparations. Schachter et al. and Nyquist and colleagues have already provided experimental techniques for the isolation of Golgi fractions of good purity from rodent pachytene spermatocytes and spermatids. Ample opportunity exists, then, for a detailed analysis of the number, specificity, and localization of FT enzymes during mammalian spermatogenesis. All available data imply that these enzymes will prove to be vital components in the differentiation of cells within the seminiferous epithelium.

Animals↗
PubMed1986

N-linked glycoprotein biosynthesis in the developing mouse embryo.

We have developed microenzymic assays that have, for the first time, enabled analysis of several enzymes in the pathway for N-linked glycoprotein biosynthesis in pre- and peri-implantation mouse embryos. The in vitro activities of the glycosyl transferases responsible for the formation of N-acetylglucosaminylpyrophosphoryldolichol,N, N'-diacetyl-chitobiosylpyrophosphoryldolichol, mannosylphosphoryldolichol, and glucosylphosphoryldolichol were found to decrease after fertilization before increasing significantly at the blastocyst stage, a stage that was also found to be highly sensitive to the glycosylation inhibitor, tunicamycin. The observed elevation in the activities of these enzymes in blastocysts still occurred when ebbryos were cultured in alpha-amanitin, indicating that de novo mRNA synthesis is unnecessary for the observed increase in their activities. Thus, an elevated capacity for N-glycosylation exists at the blastocyst stage, a time when dramatic increases in cell-cell interactions are known to occur.

Amanitins↗
PubMed1986

Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts.

The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrowth on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contrast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in vitro implantation.

Animals↗
PubMed1986

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata.

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.

Acid Phosphatase↗
PubMed1986

The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: evidence for the involvement of the cell recognition tripeptide Arg-Gly-Asp.

Differentiation of the nonadherent trophectoderm cells of the mammalian embryo into attachment-competent trophoblast cells appears to be a prerequisite to invasion of the uterine stroma. To investigate the molecular basis of trophoblast differentiation free of maternal environmental constraints, we used a model system in which attachment and outgrowth of trophoblast cells occurs in vitro. Recently, it was found that either fibronectin or laminin, both of which are extracellular matrix glycoproteins of the uterine stroma, will support trophoblast outgrowth in vitro. In this study we report that the outgrowth of blastocysts on fibronectin-coated dishes is inhibited in a dose-dependent manner by the presence of a hexapeptide containing the sequence Arg-Gly-Asp, which has been shown previously to be recognized by the fibronectin receptor. This peptide had no effect on laminin-mediated trophoblast outgrowth, suggesting that the trophoblasts contain different cell surface receptors for fibronectin and laminin. Trophoblast attachment and limited outgrowth also could be obtained on dishes to which the hexapeptide Gly-Arg-Gly-Asp-Ser-Pro was coupled. Under these conditions, however, outward migration of the trophoblast cells appeared to be reduced. Vitronectin, another adhesion molecule that apparently binds to cells via a cell surface receptor that recognizes Arg-Gly-Asp sequences, also was capable of supporting trophoblast outgrowth. These findings suggest that differentiation of cells of the trophectoderm into trophoblast cells with an invasive phenotype may involve the production of cell surface receptors for fibronectin and possibly for other proteins that contain the Arg-Gly-Asp recognition sequence.

Amino Acid Sequence↗
PubMed1982

Immunochemical analysis of discoidins I and II at the cell surface in wild type and aggregation-defective mutants of Dictyostelium discoideum.

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-state specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with 125I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by preadsorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified 125I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.

Carrier Proteins↗
PubMed1982

Discoidins I and II: common and unique regions on two lectins implicated in cell--cell cohesion in Dictyostelium discoideum.

As Dictyostelium discoideum amoebae differentiate from the noncohesive to the mutually cohesive state, they synthesize two galactose-binding lectins--discoidins I and II--which have been implicated as obligatory components of the morphogenetic cell-cell recognition and cohesion system. These proteins have been shown to have similar amino acid compositions and subunit Mr and overlapping but distinct carbohydrate recognition specificities. We have performed extensive immunochemical and biochemical analyses to study the structural relationships between these two molecules and to eventually identify structural and functional domains. Antisera raised against highly purified preparations of discoidin I and discoidin II were tested for their reactivities against each protein by both immunoprecipitation and double diffusion analyses. The patterns of crossreactivity indicated the presence of shared as well as unique antigenic determinants. This interpretation was supported by two-dimensional thin-layer peptide map analysis and by studies with purified peptides. Of approximately 10-12 peptides observed after exhaustive tryptic digestion of each radioiodinated lectin, 3 appeared to be common to both. These putative common peptides were purified, and the corresponding peptides from discoidins I and II were found to behave identically by two-dimensional thin-layer analysis, gel filtration, and susceptibility to chymotrypsin. The finding of common and unique regions in discoidins I and II suggests analogies with other families of recognition proteins and may have important functional implications for these cell-cell recognition molecules.

Carrier Proteins↗