Hymenolepis diminuta: lack of sulphate activation and sulphotransferase activity

There is no evidence that Hymenolepis diminuta can carry out sulphoconjugation reactions. Neither whole worms nor worm extracts were able to sulphate 4-methylumbelliferone. No sulphotransferase activity could be demonstrated in H. diminuta using a variety of substrates, nor was H. diminuta capable of synthesising the sulphate donor 3'-phosphoadenosine-5'-phosphosulphate from ATP and inorganic sulphate. Possible alternative sources of active sulphate in this parasite are discussed.     

Hormonal effects on the activities of glycosidases in the endometrium of rabbit uterus

The activities of several glycosidases in the lysosomal fraction of the uterine endometrium of rabbit were measured using 4-MU-glycosides as substrates. The specific activity of beta-N-acetylglucosaminidase was the highest, which was followed by beta-galactosidase, beta-glucuronidase, and alpha-galactosidase in this order. beta-Glucosidase had the lowest activity among the glycosidases examined. In order to examine the hormonal effects on these glycosidases, the lysosomal fractions were prepared from the uterine endometrium of the control, estrogen-treated, and progesterone-treated rabbits. In all glycosidases examined, except for beta-glucosidase, the specific activity was highest in the lysosome obtained from estrogen-treated rabbit. The specific activity in the lysosome from the progesterone-treated rabbit was between that from the estrogen-treated rabbit and that from control. Hormonal treatments, however, affected neither pH optimum curves nor isozyme patterns of these glycosidases.     

Biosynthesis of sulfated glycoprotein in the endometrium of rabbit uterus.

The endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-d[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25m sucrose, and the insoluble residue (R-Fr) was separate. The supernatant at 8,500Xg for 10 min of the homogenate was subjected to subcelular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (g-fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr. In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-ACETYL-D[1-3H]glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.     

Effects of hyaluronate and heparan sulphate on collagen-fibronectin interactions

Interactions of fibronectin and glycosaminoglycans and the involvement of heparan sulphate and hyaluronate in fibronectin-collagen interactions have been studied by affinity chromatography. Partially periodate-oxidized glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. The elution of fibronectin was performed by using increasing concentrations of NaCl. Of the copolymeric glycosaminoglycans, heparin and self-associating heparan sulphates display the highest affinity towards fibronectin while hyaluronic acid and chondroitin 6-sulphate do not bind fibronectin. Competitive release experiments suggest the existence of common binding sites for copolymeric glycosaminoglycans on the fibronectin backbone. Heparan sulphate favours the formation of collagen-fibronectin complexes at low molarity, while hyaluronate is ineffective at low concentrations and prevents the formation of complexes when present at concentrations > 1 mg ml^?1. It is suggested that heparan sulphate promotes the formation of complexes which bind with fibronectin thus producing steric changes that increase the affinity for collagen, while hyaluronate prevents the binding of fibronectin to collagen by a steric exclusion mechanism.     

Effects of female sex hormones on the activity of serum hyaluronidase

The activities of serum hyaluronidase from female rats were measured during pregnancy. In pregnant female rats, the activity of serum hyaluronidase was found to increase initially and to fall to a minimum by the last day of the gestation period, but the activity of the enzyme increased after delivery and was similar to normals at 21 days post-partum. The activity of hyaluronidase obtained from bovine leucocytes was significantly increased when leucocytes were incubated with various concentrations of progesterone. It is suggested that female sex hormones affect lysosomal membranes making them lyse more readily and hence release hyaluronidase.     

Effect of ovarian steroid hormones on superficial activator calcium in the rabbit uterus.

Effect of oestradiol (E2) and progesterone (P) on activator calcium binding was studied in the rabbit uterus. Superficial Ca-binding of isolated uterine strips was characterized by determining the rate of loss of isometric tension evoked by low (2.4 V/cm) field strength electrical stimulation in Ca-free Krebs solution. Intramuscular injection of 10 mg P increased superficial Ca-binding significantly in the postpartum and E2-treated virgin uterus with a latency period of 8--12 hours. Bilateral ovariectomy on the 25th day of pregnancy decreased superficial Ca-binding progressively, which could be avoided by P-substitution. 72 hours after ovariectomy P-treatment failed to increase Ca-binding. Local application of cycloheximide increased Ca-binding in the E2-treated virgin uterus. The results suggest that a high P-level plays an important role in the induction and maintenance of a strong binding of superficial activator calcium in the rabbit uterus. Progressive disappearance of the strong binding near term and after bilateral ovariectomy correlates well with P-withdrawl in this species.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze fracture techniques have been used to study the apical membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. In the ovariectomized but otherwise untreated rat, intramembranous particles (IMPs) occur at a density of 1395±122 per μm²; they appeared spherical and uniformly distributed. After 3 days treatment with estrogen, no change in appearance or density was found, but 3 days of progesterone treatment produced a significant increase in IMP density to 1622±104. Treatment with progesterone, with an additional dose of estrogen on day 3, is known to produce an epithelium receptive to the implanting blastocyst. In these conditions, the IMP density rose to 3818±337; rod-shaped particles and aggregations of IMPs were seen, and some particle arrays resembling gap junctions, in addition to the isolated spherical particles.     

Effects of ovarian hormones on cell membranes in the rat uterus

Histochemical techniques, including radioisotope histochemistry, have been used to investigate the nature of the surface carbohydrates at the apex of cells of the luminal epithelium of the rat uterus under various hormonal conditions. Binding of ruthenium red was quantitatively similar in ovariectomized rats without further treatment and in those given three daily injections of progesterone. Ruthenium red binding was significantly lower after 3 days treatment with estradiol, and also after 3 days treatment with progesterone with an additional dose of estradiol on day 3, a regime known to produce an epithelium receptive to the implanting blastocyst. Binding of concanavalin A (con A), whether studied by electron microscope histochemistry after incubation of tissue with con A-horseradish peroxidase, or by light microscope autoradiography after incubation with³H-con A, was not statistically different in any of the four groups of rats. The results with ruthenium red show a reduction in net negative charge of the carbohydrates on the apical cell membrane in conditions permitting implantation: this change is not due to variations in the amounts of the neutral carbohydrates, mannose and glucose, as demonstrated by con A.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF2 alpha the treatment with progesterone (4 mg X day-1, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol-17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE2, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE2 catabolism into 15-keto-PGE2 as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF_2α the treatment with progesterone (4 mg.day⁻¹, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol- 17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE₂, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE₂ catabolism into 15-keto- PGE₂ as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Phospholipase A2 activity in the rat uterus: Modulation by steroid hormones

Phospholipase A2 (PLA2), an enzyme which provides free arachidonic acid for the synthesis of prostaglandins (PG), has been studied in the rat uterus under various experimental conditions. Uterine PLA2 activity increased 14 fold in hypophysectomized rats implanted with Silastic capsules containing estradiol-17β as compared to those treated with oil vehicle. Dexamethasone treatment reduced the PLA2 activity induced by estrogen by 78%. Hypophysectomized animals treated with progesterone (2mg/day) for 5 days had low levels of uterine PLA2 activity but a single injection of estradiol (10ug/rat) given 24 h after the last injection of progesterone increased activity 5 fold within 12 h. Administration of the protein synthesis inhibitor cycloheximide in the rats treated with progesterone, before and after injection of estradiol, prevented the stimulating action of the estrogen on PLA2 activity. If the estrogen was given at the time of the last injection of progesterone, PLA2 activity did not increase until 24 h later and the level was much less than when progesterone was absent. The results are consistent with the view that estrogen stimulates uterine prostaglandin production because of its effect upon PLA2; this effect can be greatly reduced by a glucocorticoid. Progesterone may modulate the PLA2 stimulating effect of estrogen in order to direct the production of specific PGs by regulating the amount of arachidonic acid available for PG synthetase.     

Hormonal effects on the biosynthesis of sulfated glycoprotein in a microsomal fraction of the endometrium of rabbit uterus

A crude microsomal fraction (M-Fr) was separated from the endometrial scrapings of uteri of ovariectomized rabbits with or without hormonal treatment. The effects of estrogen and progesterone on the incorporation into M-Fr of L-[U-14C]-fucose and N-acetyl-D-[6-3H]-glucosamine from their nucleotides were investigated. Estrogen increased the incorporation of these sugars, whereas progesterone suppressed this effect. The results of fractionation on a DEAE-Sephadex A-25 (Cl- form) column of the isotope-labelled complex saccharide mixtures, obtained by pronase digestion of the incubation mixtures, indicated that biosynthesis of sulfated glycoprotein was most sensitive to the hormones among the complex saccharides in M-Fr. Thus, a hormonal effects on the biosynthesis of sulfated glycoprotein in the endometrium of ovariectomized rabbit has been unambiguously confirmed at the microsomal level.     

Fluctuations in arylsulphatase activity in the rabbit endometrium during the sexual cycle

Summary Histochemical and biochemical studies were performed to verify the presence of arylsulphatase A (ASA) and B (ASB) in the rabbit uterus. Fluctuations in the activity of these sulphatases during the sexual cycle were also studied. Some structural and functional properties of purified ASA were determined. The results indicate that arylsulphatases are active in the endometrium during both the estrogenic and progesteronic phases. The activity of ASA was much more intense than that of ASB; it increased during estrus and decreased during the post-ovulatory phase. ASB activity, however, decreased during estrus and increased during the post-ovulatory phase. The significance of these fluctuations is discussed in relation to the action of sexual hormones and physiological substrates of arylsulphatases.