- Email: [email protected]
Hormonal effect on glycoproteins and glycosaminoglycans in rabbit uteri
ARCHIVES
OF
BIOCHEMISTRY
Hormonal
Effect
AND
BIOPHYSICS
166, 397-403 (1973)
on Glycoproteins
and
Rabbit MASAHIKO Department
of Biochemistry,
END0 Tohoku
Glycosaminoglycans
in
Uteri”’
AND
ZENSAKU
University
YOSIZAWA
School of Medicine,
Sendai, Japan
Received September 27, 1972 The effect of the female hormones on glycoproteins and glycosaminoglycans in uteri has been studied. The uteri taken from the ovariectomized rabbits treated with estrogen, estrogen plus progesterone, and sham administration (control) were incubated in vitro with [II-%]glucose. Subsequently, the tissues were digested extensively with pronaae, yielding crude glycan fractions. The amount and radioactivity of the crude glycan fraction increased by the treatment with estrogen, but reduced to certain level with progesterone. Separation of glycoproteins and glycosaminoglycans was achieved by stepwise elution from Dowex 1 (X2, chloride form) with increasing concentration of NaCl. The yield and radioactivity, together with the results of chemical, enzymatic, and electrophoretic studies on the resulting fractions indicated that the metabolism of a slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulfated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulfate were stimulated remarkably with estrogen, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was, however, found to be different from each other. It was noticed that sulfated glycoproteins were the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of a neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone.
The effects of estrogen and progesterone on the uterine glycoproteins and glycosaminoglycans have been studied by many investigators (l-l 1). Little is known, however, about the effects of these hormones on the metabolism of each glycoand glycosaminoglycan in the protein uterus. In order to elucidate the hormonal control mechanisms on the metabolism of glycoproteins and glycosaminoglycans in
the uterus, ovariectomized rabbits were treated with estradiol and progesterone. The uteri were incubated in vitro with [U-14C]glucose. Subsequently, sugar nucleotides, glycoproteins, and glycosaminoglycans were isolated from the incubation mixture. The results of the study on the sugar nucleotides, the precursors of the were reported in the macromolecules, previous paper (12). This paper deals with the results of the study on glycoproteins and glycosaminoglycans.
1 Abbreviations used in text: ES, uteri from estrogen-treated rabbits; PG, uteri from estrogen-progesterone-treated rabbits; C, uteri from control rabbits; EDTA, disodium ethylenediamine tetraacetate; Fr, fraction. 2 This work has been supported by grants from The Population Council, New York.
MATERIALS
397 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Materials. Pronase P was purchased from Kaken Kagaku Co. Ltd., Tokyo. Chondroitinase ABC from Proteus vulgaris (13), chondroitinase AC from Flavobacterium heparinum (13), hyalu-
398
END0
AND YOSIZAWA
hyalurolyticus (14), ronidase from Streptomyces chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate were supplied by Seikagaku Kogyo Co. Ltd., Tokyo. Heparinase from heparinadapted F. heparinum was prepared by the procedure of Linker and Hovingh (15). Amylase was purchased from Sigma Chemical Co., St. Louis. Heparan sulfate was obtained from Japan Upjohn Co. Ltd., Tokyo. Heparin was purchased from Taiyo Fishery Co. Ltd., Yokosuka. Dermatan sulfate and hyaluronic acid were prepared from human umbilical cord according to the method of Danishefsky and Bella (16). Cellulose acetate membrane, Separax, was purchased from Joko Sangyo Co. Ltd., Tokyo. [U-14C]glucose, estradiol benzoate, and progesterone were the same materials reported previously (12). Other materials were from a commercial source. Animals and incubation. Procedures for the hormonal treatment of ovariectomized rabbits and for the incubation of uteri with [U-l*C]glucose were described previously (12). Proteolytic digestion. The ethanolic suspension of the incubation mixture was centrifuged and the sediment was washed twice with absolute ethanol and then dried over CaClt in uacuo. The dried powder (1 g) was suspended in 260 ml of 0.1 M Tris-HCl buffer (pH 8.0), and incubated with 100 mg trypsin at 37°C for 12 hr in the presence of small amounts of benzoic acid and toluene. Subsequently, to the incubation mixture was added CaClz to give a final concentration of 0.01 M. One hundred milligrams each of pronase P were added every 12 hr during the incubation. The pH of the incubation mixture was maintained at 8.0 with 1 M NaOH during proteolysis. The result of the free amino acid analysis by the method of Yemm and Cocking (17) indicated that the digestion was almost completed after approximately 4 days. Thereafter, to the incubation mixture was added 5Oa/, aqueous trichloroacetic acid at 4°C to give a final concentration of 7yo. The precipitate was removed by centrifugation and the supernatant fluid was dialyzed in Visking tubings against several changes of 10 liters each of distilled water for 2 days. The nondialyzable fraction was concentrated to 10 ml under reduced pressure. To the concentrate was added the same volume of 0.2 M Tris-NC1 buffer (pH 8.0). Subsequently, pronase digestion was repeated by the similar procedures using one-fifth of the enzyme, followed by similar treatments as above. The concentrated nondialyzable fraction was lyophilized, yielding the crude glycan fraction. Fractionation of the crude glycan fraction. Fractionation was carried out according to the procedure of Schiller et al. (18). An aqueous solution of the crude glycan fraction (4060 mg
in 3 ml) was applied to a column (1 X 60 cm) of Dowex 1 X2, 200400 mesh, chloride form, which was subsequently washed with 200 ml of water. Stepwise elution was carried out with 200-ml batches each of NaCl solutions of increasing molarity from 0.1 to 2.0 M, as shown in Fig. 1 and Table II. The effluent of each step was concentrated in vacua to a small volume and then dialyzed in Visking tubings against several changes of 2 liters each of distilled water for 2 days. Subsequently, the desalted fraction was concentrated and then lyophilized. Electrophoresis. Paper electrophoresis was carried out on Toyo filter paper No. 51 (17 X 40 cm) at 70 V/cm for 5 hr in 0.1 M ammonium acetate buffer (pH 4.3), and at 60 V/cm for 6 hr in formic acid-pyridine buffer (pH 3.0) (19). Electrophoresis on cellulose acetate membrane, Separax (6 X 22 cm), was carried out at 1 mA/cm for 30 min in formic acid-pyridine buffer (pH 3.0), and at 1 mA/cm for 3 hr in 0.2 M calcium acetate (20). The substances were visualized by staining with toluidine blue (0.05yc in 7Ooj, ethanol containing 1% acetic acid) and by periodic acid-Schiff (PAS) reaction. Determination of constituents. Descending paper chromatography on the hydrolyzates with 1 M HCl for 2 hr and 4 M HCl for 4 or 15 hr at 100°C was carried out on Toyo filter paper No. 51 with n-butanol-pyridine-water (6:4:3, by volume) solvent and with ethylacetate-pyridine-n-butanol-n-butyric acid-water (10: 10:5: 1:5, by volume) solvent at room temperature. Alkaline silver reagent was used for staining. Hexosamine was determined by the method of Garde11 (21), uranic acid by the modified carbazole-HzSO4 method (22), hexose by the phenol-H804 method (23), and sulfate by the procedure of Antonopoulos (24). Sialic acid was checked by the method of Warren (25) after hydrolysis with 0.05 M HzS04 for 1 hr at 80°C. Enzymatic digestion. The digestibility of samples with chondroitinase ABC and chondroitinase AC was checked qualitatively by the procedures of Yamagata et al. (13) and Saito et al. (26), while that with hyaluronidase and heparinase by those of Ohya and Kaneko (14) and Linker and Hovingh (15), respectively. Subsequently, the digestion products were checked by electrophoresis on cellulose acetate membrane. Determination of radioactivity. Radioactivity of samples in solution was determined by the methods described previously (12). In addition, autoradiography was carried out by exposure of the [I%] compounds on the paper electrophoretograms and on the paper chromatograms to Sakura X-ray film, industrial type N.
GLYCOPROTEINS
AND
TABLE YIELD
AND RAUIOACTIVITY ESTROGEN-TREATED
to the control
I
OF THE CRUDE GLYCAN FRACTIONS OBTAINED (ES), AND ESTROGEN-PROGESTERONE-TREATED
Yield mg/one animal mg/g dry tissue Radioactivity cpm X W/one animal cpm X 103/mg crude glycan 0 Ratio
399
GLYCOSAMINOGLYCANS
FROM
(PC)
CONTROL RABBITS
(C),
C
ES
PG
4.50 (l)a 10.47 (1)
30.35 (6.7)0 21.99 (2.1)
21.43 (4.8)0 14.01 (1.3)
0.79 (1) 1.76 (1)
17.40 (22.0) 5.73 (3.3)
7.10 (9.0) 3.31 (1.9)
value.
OBX 001
FIG. 1. Elution diagrams obtained by Dowex 1 column chromatography from the crude glycan fractions of control (C), estrogen-treated (ES), and estrogen-progesterone-treated (PG) rabbits. An aqueous solution of the crude glycan fraction (40-60 mg in 3 ml) was applied to a column (1 X 60 cm) of Dowex 1 X2, 200-400 mesh, chloride form, which was subsequently washed with water. Stepwise elution was carried out with NaCl as described in the text.
RESULTS
The yield and radioactivity of the crude glycan fractions obtained from sham administrated (control) (C), estrogen-treated (ES), and estrogen-progesterone-treated (PG) rabbits are shown in Table I. As can be seen in the table, the amount and radioactivity of the crude glycan fraction increased remarkably by the treatments of estrogen, but reduced to certain level with progesterone. Elution diagrams obtained by the Dowex 1 column chromatography of the crude glycan fractions are shown in Fig. 1. Recovery and relative radioact.ivity of t,he
resulting fractions are listed in Tables II and III, respectively. The data showed that the relative amount (mg/g dry tissue) and the relative radioactivity (cpm/lOO wg dry matter) of most of the fractions increased in ES, but decreased in PG. Of these fractions, Hz0 Fr, 0.3 M Fr, 0.5 M Fr 0.7 M Fr, 0.9 M Fr, 1.0 M Fr, 1.25 M Fr, and 1.5 M Fr were found to be very sensitive to the hormonal treatments. On the other hand, the relative amounts of 0.1 M Fr, 1.75 M Fr, and 2.0 M Fr increased in ES, but did not reduce in PG. Most of the radioactivity was found in Hz0 Fr (70-80% of the total) and in the fractions eluted with NaCl at lower concentrations. However, the relative radioTABLE
II
RECOVERY” OF THE FRACTIONS OBTAINED BY DOWEX 1 COLUMN CHROMATOGRAPHY FROM THE CRUDE GLYCAN FRACTIONS OF CONTROL (C), ESTROGEN-TREATED (ES), AND ESTROGENPROGESTERONE-TREATED (PG) RABBITS
Fraction 1120 Fr 0.1 M Fr 0.3 M Fr 0.5 M Fr 0.7 u Fr 0.9 M Fr l.OMFr 1.25 M Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
C 1.72 1.45 1.47 0.68 0.47 0.47 0.30 0.43 0.85 0.60 0.43
ES (l)b (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)
i 4.78 2.01 2.64 1.36 1.05 1.87 1.36 1.43 2.17 1.16 0.98
(2.8)* (1.4) (1.8) (2.0) (2.2) (4.0) (4.5) (3.3) (2.6) (1.9) (2.3)
PG 2.68 1.94 1.51 0.82 0.58 0.44 0.41 0.58 1.79 1.17 1.00
(l.6)b (1.3) (1.0) (1.2) (1.2) (0.9) (1.4) (1.3) (2.1) (2.0) (2.3)
a Recovery is expressed as mg/g dry tissue. b Ratio to the control value.
400
END0
AND
TABLE III RELATIVE RADIOACTIVITY~ OF THE FRACTIONS OBTAINED BY DOWEX 1 COLUMN CHROMATOGRAPHYFROMTHECRUDEGLYCANFR.~CTIONS OF CONTROL (C), ESTROGEN-TREATED (ES), AND ESTROGEN-PROGESTERONE-TREATED (PG) RABBITS
-
Fraction HtO Fr 0.1 M Fr 0.3 M Fr 0.5 M Fr 0.7 M Fr 0.9 M Fr 1.0 M Fr 1.25 M Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
_-
C
ES
PG
__ 1080 122 83 38 31 10 5 10 1 1 9
(1)b (1) (1) (1) (1) (1) 0) (1)
-
2412 870 463 338 160 118 73 84 35 16 14
(2.2)b (7.1) (5.6) (8.9) (5.2) (11.8) (14.6) (8.4)
i
1735 567 333 201 53 60 44 67 29 15 13
a Relative radioactivity is expressed 100 pg dry matter of each fraction. b Ratio to the control value.
(1.6)b (4.6) (4.0) (5.3) (1.7) (6.0) (8.8) (6.7)
as cpm/
activity of all the fractions increased remarkably in ES, but decreased to certain level in PG. The depression of the relative radioactivity in PG was the most significant in 0.7 M Fr. Since the electrophoretograms on paper and cellulose acetate membrane of the corresponding fractions of C, ES, and PG showed similar profiles, a representative one is shown in Fig. 2. As can be seen in the figure, 0.1 M Fr and 0.3 RI Fr separated into two bands, while other fractions gave one band. Of these fractions, Hz0 Fr, 0.1 M Fr, 0.3 M Fr, 0.7 M Fr, and 0.9 M Fr reacted positively to PAS staining. Fr 0.7 M, 0.9 M Fr, 1.0 M Fr, 1.25 nr Fr, 1.5 M Fr, 1.75 M Fr, and 2.0 M Fr were stained metachromatically with toluidine blue, whereas 0.5 M Fr orthochromatically with the same dye. On the other hand, Hz0 Fr, 0.1 RI Fr, 0.3 M Fr, 0.5 M Fr, 0.7 M Fr, and 0.9 M Fr were shown to be radioactive by autoradiography on the paper electrophoretograms. The mobilities of the bands suggested that Hz0 Fr, 0.5 M Fr, 1.25 M Fr, and 1.5 M Fr contained glycogen, hyaluronic acid, heparan sulfate, and chondroitin 4-sulfate,
YOSIZAWA
respectively. Fr 0.7 M and 0.9 M Fr, which migrated between hyaluronic acid and keratan sulfate, showed similar mobilities to those of sulfated glycoproteins reported previously (27). The mobility of the major component of 0.3 M Fr was similar to that of sialic acid-containing glycoprotein reported previously (27). Fr 1.0 M migrated between hyaluronic acid and keratan sulfate. Mobilities of 1.75 M Fr and 2.0 M Fr were slightly larger than that of chondroitin 4-sulfate. Although chondroitin 4-sulfate and chondroitin 6-sulfate showed similar mobility in formic acid-pyridine buffer, they were separated in 0.2 M calcium acetate by electrophoresis on cellulose acetate membrane (20). Because of the paucity of the isotopelabeled samples, the constituent monosaccharides of these fractions were checked by paper chromatography. Since similar results were obtained on the corresponding fractions of C, ES, and PG, a representative one is shown in Table IV. Glucose was found in Hz0 Fr, 0.1 M Fr, and 0.3 M Fr. Fr 0.1 $1, 0.3 M Fr, 0.7 M Fr, and 0.9 M Fr contained the constituent sugars of glycoprotein, while 0.5 M Fr, 1.0~ Fr, 1.25 M Fr, 1.5 M Fr, 1.75 M Fr and 2.0 M Fr those of glycosaminoglycans. The molar ratios of total hexose, total uranic acid, and sulfate to total hexosamine of the fractions obtained from the estrogen-treated sample are listed in Table
40 20
c
0
-0 ---
-----
---_
--
W 01 03 05 07 09 1.01’25 15 175 20 Gly HA UrS DS HS KS Hep
FIG. 2. Tracing of an electrophoretogram on cellulose acetate membrane of the fractions obtained by the Dowex 1 column chromatography of the crude glycan fraction from the est,rogentreated rabbits. Electrophoresis was carried out in formic acid-pyridine buffer (pH 3.0) at 1 mA/ cm for 3 hr, staining by PAS reaction and with toluidine blue. HzO, 0.1, . . . . . 2.0: Hz0 Fr, 0.1 M Fr, . . . . . 2.0 M Fr; Gly, glycogen; HA, hyaluronic acid; ChS, chondroitin 4-sulfate; DS, dermatan sulfate; HS, heparan sulfate; KS, keratan sulfate; Hep, heparin.
GLYCOPROTEINS
AND
V. Approximately equimolar total hexose or total uranic acid to total hexosamine was found in most of these fractions. The fractions eluted with NaCl at the concentrations from 0.7 nr to 2.0 M contained sulfate. The molar ratio of sulfate to total hexosaTABLE
mine increased with the increasing molarity of NaCl. In order to characterize these fractions qualitatively, the digestibility with enzymes was checked. A preliminary result showed that H20 Fr, 0.5 M Fr, 1.5 M Fr, 1.75 RI Fr were digested well with amylase, hyaluronidase, chondroitinase ABC, and chondroitinase AC, respectively, while a small amount of 1.5 M Fr remained intact after digestion with chondroitinase AC. -4lthough a certain amount of 1.0 3% Fr, a small amount of 2.0 nr Fr, and a large amount of 1.25 RI Fr were not digested with chondroitinase ABC, they were digested with heparinase. The above observations indicated that Hz0 Fr was glycogen; 0.1 RI Fr, glycopeptide plus glycogen; 0.3 11 Fr, slightly acidic glycopeptide plus a small amount of glycogen; 0.5 M Fr, hyaluronic acid; 0.7 M Fr and 0.9 M Fr, sulfated glycopeptides plus small amount’s of glycosaminoglycans; 1.0 hi Fr, low-sulfated chondroitin sulfate plus heparan sulfate; 1.25 M Fr, heparan sulfate plus a small amount of chondroit’in sulfate; 1.5 M Fr, chondroitin 4-sulfate plus a small amount of dermatan sulfate; 1.75 M Fr, oversulfated chondroitin sulfate; 2.0 M Fr, oversulfated chondroitin sulfate plus a small amount of hcparin. Radioactivit’g was found in all t’he fractions obtained by the Dowex 1 column chromatography of the crude glycan frac-
IV
~~o~os.kcc~a~~~~s DETECTED ON THE FRACTIONS OBTAINED BY DOTVEX 1 COLUMN CHROMATOGRAPHY OFTHE CRUDE GLYCAN FRACTION OF THE ESTROGEN-TRE.~TED RABBITS
-
Monosaccharide* Fraction
Major
Hz0 Fr 0.1 M Fr 0.3 M 0.5 M 0.7 M 0.9 M 1 .O M
Fr Fr Fr Fr Fr
1.25 Y Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
Minor
Glc Glc, Gal, GlcN, GalN Gal, GlcN, GalN GlcN, GlcUA Gal, GlcN, GalN Gal, GlcN, GalN GlcN, GalN, GlcUA GlcN, GlcUA GalN, GlcUA GalN, GlcUA GalN, GlcUA
Man,
Fuc
Glc, Fuc, SAb GlcUA, GlcUA,
401
GLYCOSAMINOGLYCANS
Fuc, SA Fuc, SA
GalN IdUA GlcN
0 Detected by paper chromatography on the hydrolyzates with 1 in HCl for 2 hr and 4 M HCI for 4 or 15 hr. b Sialic acid was checked by the method of Warren (25). TABLE
V
MOLAR RATIO OF THE COKSTITUEXTP OF THE FRACTIONS OBTAINED UT DOWEX~ CHRO~ATOGR.~PHYOFTHECRUDEGLYCANFRACTIONFROMTHEESTROGEN-TREATEDR.~BBITS Fraction
Constituent
Hexose Hexosamine Uranic
0.1
0.3
2.9
1.4
acid
Hexosamine
0.5
1.0
0.2 acid, and total
hexosamine
(M) 1.0
1.25
1.5
1.75
2.0
1.0
1.2
0.9
1.0
1.1
0.4
0.7
1.0
1.2
1.4
1.1
1.1
Sulfate Hexosamine a Total hexose, total uranic and glucosamine, respectively.
0.7
0.9
COLUMN
0.3
were expressed
as galactose,
glucuronic
acid,
402
END0
AND YOSIZAWA
tions. The relative radioactivity of each fraction increased in ES, but was restored to a certain level in PG. However, the value in PG was significantly higher than that in C. Moreover, the degree of the change of the relative radioactivity by the hormonal treatments was found to be different from each other. Although the radioactivity of the fractions eluted with water and with NaCl at concentrations from 0.1 M to 0.9 M were detectable by autoradiography on paper electrophoretograms, the radioactivity of glucose, galactose, and glucosamine in Hz0 Fr, 0.1 hf Fr, and 0.3 M Fr was noticed by autoradiography on paper chromatograms only after acid hydrolysis. DISCUSSION
The results of the present study on the fractions obtained by the Dowex 1 column chromatography of the crude glycan fractions indicated that the uterine glycoproteins and glycosaminoglycans might be divided into two groups in respect to the response to hormonal treatments. The stimulation of the metabolism with estrogen of glycogen, slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulfated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulfate was remarkable, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was found to be different from each other. Of these substances, sulfated glycoproteins were noticed to be the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone. The hormone-sensitive uterine glycoproteins and glycosaminoglycans might play an important role for reproduction. Radioactivity was found in all the fractions obtained by Dowex 1 column chromatography from the crude glycan fractions. The relative radioactivities of all the fractions increased remarkably in ES, but were restored to certain level in PG. However, the values in PG were significantly higher than those in C, indicating that higher metabolic rates induced by estrogen of
these substances remained at a certain level in PG. Radioactivity of glucose, galactose, and glucosamine in glycogen and glycopeptides (Hz0 Fr, 0.1 M Fr, and 0.3 M Fr) was detectable by autoradiography on paper chromatograms only after acid hydrolysis. The radioactivity of the other constituent sugars seemed to be too small to detect by the present technique. The low level of radioactivity incorporated into glycoproteins and glycosaminoglycans might be because of the low metabolic rate of these substances under the conditions of the present study. The presence of hyaluronic acid, chondroitin sulfate, heparan sulfate, and other glycosaminoglycans in uteri was thus far reported (l-9). Moreover, the biosynthesis of these glycosaminoglycans was shown to be stimulated with estrogen (5-S), whereas progesterone was able to block the synthesis of glycosaminoglycans (7). It was reported also that uterine glycoproteins increased with estrogen (10, II), but decreased with progesterone (11). These observations indicated that estrogen stimulated the metabolism of the uterine glycoproteins and glycosaminoglycans, but the stimulation was suppressed with progesterone. However, the effects of estrogen and progesterone on the metabolism of each glycoprotein and glycosaminoglycan have not yet been investigated comprehensively. The changes observed on most of the glycoproteins and glycosaminoglycans in the present study were similar to those reported by other investigators (j-11). In the present study, however, the more extensive fractionation and the more comprehensive characterization of the substances permitted the detection of effects of estrogen and progesterone on each glycoprotein and glycosaminoglycan, specifically on the sulfated glycoproteins in the rabbit uteri. REFERENCES 1. ZACHARIAE, F., AND THORS$E, H. (1966) in Hormones and Connective Tissue (AsboeHansen, G., ed.), p. 257, Munksgaard, Copenhagen. 2. LIKAR, I. N., LIKAR, L. J., AND TAYLOR, H. E. (1961) Nature (London) 190,118.
GLYCOPROTEINS
AND
3. LIKAR, I. N., LIKAR, L. J., AND ROBINSON, R. W. (1964) Nature (London) 203,730 4. LOEWI, G., AND CONSDEN, R. (1962) Nature (London) 196, 148. 5. IVERSEN, 0. H. (1962) Acta Pathol. Microbial. Stand. 66, 245. 6. BOSTR~M, H., AND ODEBLAD, E. (1952) Acta Endocrinol. 10, 89. 7. ZACHARIAE, F. (1958) Acta EndocrinoZ. 29,118. 8. SINOHARA, H., AND SKY-PECK, H. H. (1964) Arch. Biochem. Biophys. 106, 138. 9. HENZEL, M., SMITH, R. E., MAGOUN, R. E., AND HILL, R. (1968) Fert. Steril. 19,914. 10. DUGAN, F. A., RADHAKRISHNAMURTHY, B., RUDMAN, R. A., .~ND BERENSON, G. S. (1968) J. Endocrinol. 42, 261. 11. COPPOLA, J. A., .~ND BBLL, J. L. (1966) Steroid 6, 345. 12. ENDO, M., AND YOSIZAWA, Z. (1968) Arch. Biochem. Biophys. 137, 5%. 13. YAM.~GATA, T., SAITO, H., HABUCHI, O., AND SUZUKI, S. (1968) J. Biol. Chem. 243,1523. 14. OHYA, T., -END KANEKO, Y. (1970) Biochim. Biophys. Acta 198, 607. 15. LINKER, A., AND HOVINGH, P. (1965) J. Biol. Chem. 240, 3724.
GLYCOSAMINOGLYCANS
403
16. DANISHEFSKY, L., BND BELLA, A., JR. (1966) J. Biol. Chem. 241, 143. 17. YEMM, E. W., AND COCKING, E. C. (1955) Analyst 80, 209. 18. SCHILLER, S., SLOVER, G. A., AND DORFMAN, A. (1961) J. Biol. Chem. 236, 983. 19. MATHEWS, M. B. (1961) Biochim. Biophys. Acta 48, 402. 20. SENO, N., ANNO, K., KONDO, K., NAGASE, S., AND SAITO, S. (1970) Anal. Biochem. 37, 197. 21. GARDELL, S. (1953) Acta Chem. &and. 7, 207. 22. BITTER, T., AND MUIR, H. (1962) Anal. Biothem. 4, 330. 23. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350. Chem. 24. ANTONOPOULOS, C. A. (1962) Acta &and. 16, 1521. 25. WARREN, L. (1959) J. BioZ. Chem. 234, 1971. 26. SAITO, H., YAMAGATA, T., AND SUZUKI, S. (1968) J. BioZ. Chem. 243, 1536. 27. MATSUSHIRO, T., NEMOTO, T., ENDO, M., AND YOSIZAWA, Z. (1970) Clin. Chim. Acta 30, 645.
OF
BIOCHEMISTRY
Hormonal
Effect
AND
BIOPHYSICS
166, 397-403 (1973)
on Glycoproteins
and
Rabbit MASAHIKO Department
of Biochemistry,
END0 Tohoku
Glycosaminoglycans
in
Uteri”’
AND
ZENSAKU
University
YOSIZAWA
School of Medicine,
Sendai, Japan
Received September 27, 1972 The effect of the female hormones on glycoproteins and glycosaminoglycans in uteri has been studied. The uteri taken from the ovariectomized rabbits treated with estrogen, estrogen plus progesterone, and sham administration (control) were incubated in vitro with [II-%]glucose. Subsequently, the tissues were digested extensively with pronaae, yielding crude glycan fractions. The amount and radioactivity of the crude glycan fraction increased by the treatment with estrogen, but reduced to certain level with progesterone. Separation of glycoproteins and glycosaminoglycans was achieved by stepwise elution from Dowex 1 (X2, chloride form) with increasing concentration of NaCl. The yield and radioactivity, together with the results of chemical, enzymatic, and electrophoretic studies on the resulting fractions indicated that the metabolism of a slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulfated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulfate were stimulated remarkably with estrogen, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was, however, found to be different from each other. It was noticed that sulfated glycoproteins were the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of a neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone.
The effects of estrogen and progesterone on the uterine glycoproteins and glycosaminoglycans have been studied by many investigators (l-l 1). Little is known, however, about the effects of these hormones on the metabolism of each glycoand glycosaminoglycan in the protein uterus. In order to elucidate the hormonal control mechanisms on the metabolism of glycoproteins and glycosaminoglycans in
the uterus, ovariectomized rabbits were treated with estradiol and progesterone. The uteri were incubated in vitro with [U-14C]glucose. Subsequently, sugar nucleotides, glycoproteins, and glycosaminoglycans were isolated from the incubation mixture. The results of the study on the sugar nucleotides, the precursors of the were reported in the macromolecules, previous paper (12). This paper deals with the results of the study on glycoproteins and glycosaminoglycans.
1 Abbreviations used in text: ES, uteri from estrogen-treated rabbits; PG, uteri from estrogen-progesterone-treated rabbits; C, uteri from control rabbits; EDTA, disodium ethylenediamine tetraacetate; Fr, fraction. 2 This work has been supported by grants from The Population Council, New York.
MATERIALS
397 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Materials. Pronase P was purchased from Kaken Kagaku Co. Ltd., Tokyo. Chondroitinase ABC from Proteus vulgaris (13), chondroitinase AC from Flavobacterium heparinum (13), hyalu-
398
END0
AND YOSIZAWA
hyalurolyticus (14), ronidase from Streptomyces chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate were supplied by Seikagaku Kogyo Co. Ltd., Tokyo. Heparinase from heparinadapted F. heparinum was prepared by the procedure of Linker and Hovingh (15). Amylase was purchased from Sigma Chemical Co., St. Louis. Heparan sulfate was obtained from Japan Upjohn Co. Ltd., Tokyo. Heparin was purchased from Taiyo Fishery Co. Ltd., Yokosuka. Dermatan sulfate and hyaluronic acid were prepared from human umbilical cord according to the method of Danishefsky and Bella (16). Cellulose acetate membrane, Separax, was purchased from Joko Sangyo Co. Ltd., Tokyo. [U-14C]glucose, estradiol benzoate, and progesterone were the same materials reported previously (12). Other materials were from a commercial source. Animals and incubation. Procedures for the hormonal treatment of ovariectomized rabbits and for the incubation of uteri with [U-l*C]glucose were described previously (12). Proteolytic digestion. The ethanolic suspension of the incubation mixture was centrifuged and the sediment was washed twice with absolute ethanol and then dried over CaClt in uacuo. The dried powder (1 g) was suspended in 260 ml of 0.1 M Tris-HCl buffer (pH 8.0), and incubated with 100 mg trypsin at 37°C for 12 hr in the presence of small amounts of benzoic acid and toluene. Subsequently, to the incubation mixture was added CaClz to give a final concentration of 0.01 M. One hundred milligrams each of pronase P were added every 12 hr during the incubation. The pH of the incubation mixture was maintained at 8.0 with 1 M NaOH during proteolysis. The result of the free amino acid analysis by the method of Yemm and Cocking (17) indicated that the digestion was almost completed after approximately 4 days. Thereafter, to the incubation mixture was added 5Oa/, aqueous trichloroacetic acid at 4°C to give a final concentration of 7yo. The precipitate was removed by centrifugation and the supernatant fluid was dialyzed in Visking tubings against several changes of 10 liters each of distilled water for 2 days. The nondialyzable fraction was concentrated to 10 ml under reduced pressure. To the concentrate was added the same volume of 0.2 M Tris-NC1 buffer (pH 8.0). Subsequently, pronase digestion was repeated by the similar procedures using one-fifth of the enzyme, followed by similar treatments as above. The concentrated nondialyzable fraction was lyophilized, yielding the crude glycan fraction. Fractionation of the crude glycan fraction. Fractionation was carried out according to the procedure of Schiller et al. (18). An aqueous solution of the crude glycan fraction (4060 mg
in 3 ml) was applied to a column (1 X 60 cm) of Dowex 1 X2, 200400 mesh, chloride form, which was subsequently washed with 200 ml of water. Stepwise elution was carried out with 200-ml batches each of NaCl solutions of increasing molarity from 0.1 to 2.0 M, as shown in Fig. 1 and Table II. The effluent of each step was concentrated in vacua to a small volume and then dialyzed in Visking tubings against several changes of 2 liters each of distilled water for 2 days. Subsequently, the desalted fraction was concentrated and then lyophilized. Electrophoresis. Paper electrophoresis was carried out on Toyo filter paper No. 51 (17 X 40 cm) at 70 V/cm for 5 hr in 0.1 M ammonium acetate buffer (pH 4.3), and at 60 V/cm for 6 hr in formic acid-pyridine buffer (pH 3.0) (19). Electrophoresis on cellulose acetate membrane, Separax (6 X 22 cm), was carried out at 1 mA/cm for 30 min in formic acid-pyridine buffer (pH 3.0), and at 1 mA/cm for 3 hr in 0.2 M calcium acetate (20). The substances were visualized by staining with toluidine blue (0.05yc in 7Ooj, ethanol containing 1% acetic acid) and by periodic acid-Schiff (PAS) reaction. Determination of constituents. Descending paper chromatography on the hydrolyzates with 1 M HCl for 2 hr and 4 M HCl for 4 or 15 hr at 100°C was carried out on Toyo filter paper No. 51 with n-butanol-pyridine-water (6:4:3, by volume) solvent and with ethylacetate-pyridine-n-butanol-n-butyric acid-water (10: 10:5: 1:5, by volume) solvent at room temperature. Alkaline silver reagent was used for staining. Hexosamine was determined by the method of Garde11 (21), uranic acid by the modified carbazole-HzSO4 method (22), hexose by the phenol-H804 method (23), and sulfate by the procedure of Antonopoulos (24). Sialic acid was checked by the method of Warren (25) after hydrolysis with 0.05 M HzS04 for 1 hr at 80°C. Enzymatic digestion. The digestibility of samples with chondroitinase ABC and chondroitinase AC was checked qualitatively by the procedures of Yamagata et al. (13) and Saito et al. (26), while that with hyaluronidase and heparinase by those of Ohya and Kaneko (14) and Linker and Hovingh (15), respectively. Subsequently, the digestion products were checked by electrophoresis on cellulose acetate membrane. Determination of radioactivity. Radioactivity of samples in solution was determined by the methods described previously (12). In addition, autoradiography was carried out by exposure of the [I%] compounds on the paper electrophoretograms and on the paper chromatograms to Sakura X-ray film, industrial type N.
GLYCOPROTEINS
AND
TABLE YIELD
AND RAUIOACTIVITY ESTROGEN-TREATED
to the control
I
OF THE CRUDE GLYCAN FRACTIONS OBTAINED (ES), AND ESTROGEN-PROGESTERONE-TREATED
Yield mg/one animal mg/g dry tissue Radioactivity cpm X W/one animal cpm X 103/mg crude glycan 0 Ratio
399
GLYCOSAMINOGLYCANS
FROM
(PC)
CONTROL RABBITS
(C),
C
ES
PG
4.50 (l)a 10.47 (1)
30.35 (6.7)0 21.99 (2.1)
21.43 (4.8)0 14.01 (1.3)
0.79 (1) 1.76 (1)
17.40 (22.0) 5.73 (3.3)
7.10 (9.0) 3.31 (1.9)
value.
OBX 001
FIG. 1. Elution diagrams obtained by Dowex 1 column chromatography from the crude glycan fractions of control (C), estrogen-treated (ES), and estrogen-progesterone-treated (PG) rabbits. An aqueous solution of the crude glycan fraction (40-60 mg in 3 ml) was applied to a column (1 X 60 cm) of Dowex 1 X2, 200-400 mesh, chloride form, which was subsequently washed with water. Stepwise elution was carried out with NaCl as described in the text.
RESULTS
The yield and radioactivity of the crude glycan fractions obtained from sham administrated (control) (C), estrogen-treated (ES), and estrogen-progesterone-treated (PG) rabbits are shown in Table I. As can be seen in the table, the amount and radioactivity of the crude glycan fraction increased remarkably by the treatments of estrogen, but reduced to certain level with progesterone. Elution diagrams obtained by the Dowex 1 column chromatography of the crude glycan fractions are shown in Fig. 1. Recovery and relative radioact.ivity of t,he
resulting fractions are listed in Tables II and III, respectively. The data showed that the relative amount (mg/g dry tissue) and the relative radioactivity (cpm/lOO wg dry matter) of most of the fractions increased in ES, but decreased in PG. Of these fractions, Hz0 Fr, 0.3 M Fr, 0.5 M Fr 0.7 M Fr, 0.9 M Fr, 1.0 M Fr, 1.25 M Fr, and 1.5 M Fr were found to be very sensitive to the hormonal treatments. On the other hand, the relative amounts of 0.1 M Fr, 1.75 M Fr, and 2.0 M Fr increased in ES, but did not reduce in PG. Most of the radioactivity was found in Hz0 Fr (70-80% of the total) and in the fractions eluted with NaCl at lower concentrations. However, the relative radioTABLE
II
RECOVERY” OF THE FRACTIONS OBTAINED BY DOWEX 1 COLUMN CHROMATOGRAPHY FROM THE CRUDE GLYCAN FRACTIONS OF CONTROL (C), ESTROGEN-TREATED (ES), AND ESTROGENPROGESTERONE-TREATED (PG) RABBITS
Fraction 1120 Fr 0.1 M Fr 0.3 M Fr 0.5 M Fr 0.7 u Fr 0.9 M Fr l.OMFr 1.25 M Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
C 1.72 1.45 1.47 0.68 0.47 0.47 0.30 0.43 0.85 0.60 0.43
ES (l)b (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)
i 4.78 2.01 2.64 1.36 1.05 1.87 1.36 1.43 2.17 1.16 0.98
(2.8)* (1.4) (1.8) (2.0) (2.2) (4.0) (4.5) (3.3) (2.6) (1.9) (2.3)
PG 2.68 1.94 1.51 0.82 0.58 0.44 0.41 0.58 1.79 1.17 1.00
(l.6)b (1.3) (1.0) (1.2) (1.2) (0.9) (1.4) (1.3) (2.1) (2.0) (2.3)
a Recovery is expressed as mg/g dry tissue. b Ratio to the control value.
400
END0
AND
TABLE III RELATIVE RADIOACTIVITY~ OF THE FRACTIONS OBTAINED BY DOWEX 1 COLUMN CHROMATOGRAPHYFROMTHECRUDEGLYCANFR.~CTIONS OF CONTROL (C), ESTROGEN-TREATED (ES), AND ESTROGEN-PROGESTERONE-TREATED (PG) RABBITS
-
Fraction HtO Fr 0.1 M Fr 0.3 M Fr 0.5 M Fr 0.7 M Fr 0.9 M Fr 1.0 M Fr 1.25 M Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
_-
C
ES
PG
__ 1080 122 83 38 31 10 5 10 1 1 9
(1)b (1) (1) (1) (1) (1) 0) (1)
-
2412 870 463 338 160 118 73 84 35 16 14
(2.2)b (7.1) (5.6) (8.9) (5.2) (11.8) (14.6) (8.4)
i
1735 567 333 201 53 60 44 67 29 15 13
a Relative radioactivity is expressed 100 pg dry matter of each fraction. b Ratio to the control value.
(1.6)b (4.6) (4.0) (5.3) (1.7) (6.0) (8.8) (6.7)
as cpm/
activity of all the fractions increased remarkably in ES, but decreased to certain level in PG. The depression of the relative radioactivity in PG was the most significant in 0.7 M Fr. Since the electrophoretograms on paper and cellulose acetate membrane of the corresponding fractions of C, ES, and PG showed similar profiles, a representative one is shown in Fig. 2. As can be seen in the figure, 0.1 M Fr and 0.3 RI Fr separated into two bands, while other fractions gave one band. Of these fractions, Hz0 Fr, 0.1 M Fr, 0.3 M Fr, 0.7 M Fr, and 0.9 M Fr reacted positively to PAS staining. Fr 0.7 M, 0.9 M Fr, 1.0 M Fr, 1.25 nr Fr, 1.5 M Fr, 1.75 M Fr, and 2.0 M Fr were stained metachromatically with toluidine blue, whereas 0.5 M Fr orthochromatically with the same dye. On the other hand, Hz0 Fr, 0.1 RI Fr, 0.3 M Fr, 0.5 M Fr, 0.7 M Fr, and 0.9 M Fr were shown to be radioactive by autoradiography on the paper electrophoretograms. The mobilities of the bands suggested that Hz0 Fr, 0.5 M Fr, 1.25 M Fr, and 1.5 M Fr contained glycogen, hyaluronic acid, heparan sulfate, and chondroitin 4-sulfate,
YOSIZAWA
respectively. Fr 0.7 M and 0.9 M Fr, which migrated between hyaluronic acid and keratan sulfate, showed similar mobilities to those of sulfated glycoproteins reported previously (27). The mobility of the major component of 0.3 M Fr was similar to that of sialic acid-containing glycoprotein reported previously (27). Fr 1.0 M migrated between hyaluronic acid and keratan sulfate. Mobilities of 1.75 M Fr and 2.0 M Fr were slightly larger than that of chondroitin 4-sulfate. Although chondroitin 4-sulfate and chondroitin 6-sulfate showed similar mobility in formic acid-pyridine buffer, they were separated in 0.2 M calcium acetate by electrophoresis on cellulose acetate membrane (20). Because of the paucity of the isotopelabeled samples, the constituent monosaccharides of these fractions were checked by paper chromatography. Since similar results were obtained on the corresponding fractions of C, ES, and PG, a representative one is shown in Table IV. Glucose was found in Hz0 Fr, 0.1 M Fr, and 0.3 M Fr. Fr 0.1 $1, 0.3 M Fr, 0.7 M Fr, and 0.9 M Fr contained the constituent sugars of glycoprotein, while 0.5 M Fr, 1.0~ Fr, 1.25 M Fr, 1.5 M Fr, 1.75 M Fr and 2.0 M Fr those of glycosaminoglycans. The molar ratios of total hexose, total uranic acid, and sulfate to total hexosamine of the fractions obtained from the estrogen-treated sample are listed in Table
40 20
c
0
-0 ---
-----
---_
--
W 01 03 05 07 09 1.01’25 15 175 20 Gly HA UrS DS HS KS Hep
FIG. 2. Tracing of an electrophoretogram on cellulose acetate membrane of the fractions obtained by the Dowex 1 column chromatography of the crude glycan fraction from the est,rogentreated rabbits. Electrophoresis was carried out in formic acid-pyridine buffer (pH 3.0) at 1 mA/ cm for 3 hr, staining by PAS reaction and with toluidine blue. HzO, 0.1, . . . . . 2.0: Hz0 Fr, 0.1 M Fr, . . . . . 2.0 M Fr; Gly, glycogen; HA, hyaluronic acid; ChS, chondroitin 4-sulfate; DS, dermatan sulfate; HS, heparan sulfate; KS, keratan sulfate; Hep, heparin.
GLYCOPROTEINS
AND
V. Approximately equimolar total hexose or total uranic acid to total hexosamine was found in most of these fractions. The fractions eluted with NaCl at the concentrations from 0.7 nr to 2.0 M contained sulfate. The molar ratio of sulfate to total hexosaTABLE
mine increased with the increasing molarity of NaCl. In order to characterize these fractions qualitatively, the digestibility with enzymes was checked. A preliminary result showed that H20 Fr, 0.5 M Fr, 1.5 M Fr, 1.75 RI Fr were digested well with amylase, hyaluronidase, chondroitinase ABC, and chondroitinase AC, respectively, while a small amount of 1.5 M Fr remained intact after digestion with chondroitinase AC. -4lthough a certain amount of 1.0 3% Fr, a small amount of 2.0 nr Fr, and a large amount of 1.25 RI Fr were not digested with chondroitinase ABC, they were digested with heparinase. The above observations indicated that Hz0 Fr was glycogen; 0.1 RI Fr, glycopeptide plus glycogen; 0.3 11 Fr, slightly acidic glycopeptide plus a small amount of glycogen; 0.5 M Fr, hyaluronic acid; 0.7 M Fr and 0.9 M Fr, sulfated glycopeptides plus small amount’s of glycosaminoglycans; 1.0 hi Fr, low-sulfated chondroitin sulfate plus heparan sulfate; 1.25 M Fr, heparan sulfate plus a small amount of chondroit’in sulfate; 1.5 M Fr, chondroitin 4-sulfate plus a small amount of dermatan sulfate; 1.75 M Fr, oversulfated chondroitin sulfate; 2.0 M Fr, oversulfated chondroitin sulfate plus a small amount of hcparin. Radioactivit’g was found in all t’he fractions obtained by the Dowex 1 column chromatography of the crude glycan frac-
IV
~~o~os.kcc~a~~~~s DETECTED ON THE FRACTIONS OBTAINED BY DOTVEX 1 COLUMN CHROMATOGRAPHY OFTHE CRUDE GLYCAN FRACTION OF THE ESTROGEN-TRE.~TED RABBITS
-
Monosaccharide* Fraction
Major
Hz0 Fr 0.1 M Fr 0.3 M 0.5 M 0.7 M 0.9 M 1 .O M
Fr Fr Fr Fr Fr
1.25 Y Fr 1.5 M Fr 1.75 M Fr 2.0 M Fr
Minor
Glc Glc, Gal, GlcN, GalN Gal, GlcN, GalN GlcN, GlcUA Gal, GlcN, GalN Gal, GlcN, GalN GlcN, GalN, GlcUA GlcN, GlcUA GalN, GlcUA GalN, GlcUA GalN, GlcUA
Man,
Fuc
Glc, Fuc, SAb GlcUA, GlcUA,
401
GLYCOSAMINOGLYCANS
Fuc, SA Fuc, SA
GalN IdUA GlcN
0 Detected by paper chromatography on the hydrolyzates with 1 in HCl for 2 hr and 4 M HCI for 4 or 15 hr. b Sialic acid was checked by the method of Warren (25). TABLE
V
MOLAR RATIO OF THE COKSTITUEXTP OF THE FRACTIONS OBTAINED UT DOWEX~ CHRO~ATOGR.~PHYOFTHECRUDEGLYCANFRACTIONFROMTHEESTROGEN-TREATEDR.~BBITS Fraction
Constituent
Hexose Hexosamine Uranic
0.1
0.3
2.9
1.4
acid
Hexosamine
0.5
1.0
0.2 acid, and total
hexosamine
(M) 1.0
1.25
1.5
1.75
2.0
1.0
1.2
0.9
1.0
1.1
0.4
0.7
1.0
1.2
1.4
1.1
1.1
Sulfate Hexosamine a Total hexose, total uranic and glucosamine, respectively.
0.7
0.9
COLUMN
0.3
were expressed
as galactose,
glucuronic
acid,
402
END0
AND YOSIZAWA
tions. The relative radioactivity of each fraction increased in ES, but was restored to a certain level in PG. However, the value in PG was significantly higher than that in C. Moreover, the degree of the change of the relative radioactivity by the hormonal treatments was found to be different from each other. Although the radioactivity of the fractions eluted with water and with NaCl at concentrations from 0.1 M to 0.9 M were detectable by autoradiography on paper electrophoretograms, the radioactivity of glucose, galactose, and glucosamine in Hz0 Fr, 0.1 hf Fr, and 0.3 M Fr was noticed by autoradiography on paper chromatograms only after acid hydrolysis. DISCUSSION
The results of the present study on the fractions obtained by the Dowex 1 column chromatography of the crude glycan fractions indicated that the uterine glycoproteins and glycosaminoglycans might be divided into two groups in respect to the response to hormonal treatments. The stimulation of the metabolism with estrogen of glycogen, slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulfated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulfate was remarkable, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was found to be different from each other. Of these substances, sulfated glycoproteins were noticed to be the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone. The hormone-sensitive uterine glycoproteins and glycosaminoglycans might play an important role for reproduction. Radioactivity was found in all the fractions obtained by Dowex 1 column chromatography from the crude glycan fractions. The relative radioactivities of all the fractions increased remarkably in ES, but were restored to certain level in PG. However, the values in PG were significantly higher than those in C, indicating that higher metabolic rates induced by estrogen of
these substances remained at a certain level in PG. Radioactivity of glucose, galactose, and glucosamine in glycogen and glycopeptides (Hz0 Fr, 0.1 M Fr, and 0.3 M Fr) was detectable by autoradiography on paper chromatograms only after acid hydrolysis. The radioactivity of the other constituent sugars seemed to be too small to detect by the present technique. The low level of radioactivity incorporated into glycoproteins and glycosaminoglycans might be because of the low metabolic rate of these substances under the conditions of the present study. The presence of hyaluronic acid, chondroitin sulfate, heparan sulfate, and other glycosaminoglycans in uteri was thus far reported (l-9). Moreover, the biosynthesis of these glycosaminoglycans was shown to be stimulated with estrogen (5-S), whereas progesterone was able to block the synthesis of glycosaminoglycans (7). It was reported also that uterine glycoproteins increased with estrogen (10, II), but decreased with progesterone (11). These observations indicated that estrogen stimulated the metabolism of the uterine glycoproteins and glycosaminoglycans, but the stimulation was suppressed with progesterone. However, the effects of estrogen and progesterone on the metabolism of each glycoprotein and glycosaminoglycan have not yet been investigated comprehensively. The changes observed on most of the glycoproteins and glycosaminoglycans in the present study were similar to those reported by other investigators (j-11). In the present study, however, the more extensive fractionation and the more comprehensive characterization of the substances permitted the detection of effects of estrogen and progesterone on each glycoprotein and glycosaminoglycan, specifically on the sulfated glycoproteins in the rabbit uteri. REFERENCES 1. ZACHARIAE, F., AND THORS$E, H. (1966) in Hormones and Connective Tissue (AsboeHansen, G., ed.), p. 257, Munksgaard, Copenhagen. 2. LIKAR, I. N., LIKAR, L. J., AND TAYLOR, H. E. (1961) Nature (London) 190,118.
GLYCOPROTEINS
AND
3. LIKAR, I. N., LIKAR, L. J., AND ROBINSON, R. W. (1964) Nature (London) 203,730 4. LOEWI, G., AND CONSDEN, R. (1962) Nature (London) 196, 148. 5. IVERSEN, 0. H. (1962) Acta Pathol. Microbial. Stand. 66, 245. 6. BOSTR~M, H., AND ODEBLAD, E. (1952) Acta Endocrinol. 10, 89. 7. ZACHARIAE, F. (1958) Acta EndocrinoZ. 29,118. 8. SINOHARA, H., AND SKY-PECK, H. H. (1964) Arch. Biochem. Biophys. 106, 138. 9. HENZEL, M., SMITH, R. E., MAGOUN, R. E., AND HILL, R. (1968) Fert. Steril. 19,914. 10. DUGAN, F. A., RADHAKRISHNAMURTHY, B., RUDMAN, R. A., .~ND BERENSON, G. S. (1968) J. Endocrinol. 42, 261. 11. COPPOLA, J. A., .~ND BBLL, J. L. (1966) Steroid 6, 345. 12. ENDO, M., AND YOSIZAWA, Z. (1968) Arch. Biochem. Biophys. 137, 5%. 13. YAM.~GATA, T., SAITO, H., HABUCHI, O., AND SUZUKI, S. (1968) J. Biol. Chem. 243,1523. 14. OHYA, T., -END KANEKO, Y. (1970) Biochim. Biophys. Acta 198, 607. 15. LINKER, A., AND HOVINGH, P. (1965) J. Biol. Chem. 240, 3724.
GLYCOSAMINOGLYCANS
403
16. DANISHEFSKY, L., BND BELLA, A., JR. (1966) J. Biol. Chem. 241, 143. 17. YEMM, E. W., AND COCKING, E. C. (1955) Analyst 80, 209. 18. SCHILLER, S., SLOVER, G. A., AND DORFMAN, A. (1961) J. Biol. Chem. 236, 983. 19. MATHEWS, M. B. (1961) Biochim. Biophys. Acta 48, 402. 20. SENO, N., ANNO, K., KONDO, K., NAGASE, S., AND SAITO, S. (1970) Anal. Biochem. 37, 197. 21. GARDELL, S. (1953) Acta Chem. &and. 7, 207. 22. BITTER, T., AND MUIR, H. (1962) Anal. Biothem. 4, 330. 23. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350. Chem. 24. ANTONOPOULOS, C. A. (1962) Acta &and. 16, 1521. 25. WARREN, L. (1959) J. BioZ. Chem. 234, 1971. 26. SAITO, H., YAMAGATA, T., AND SUZUKI, S. (1968) J. BioZ. Chem. 243, 1536. 27. MATSUSHIRO, T., NEMOTO, T., ENDO, M., AND YOSIZAWA, Z. (1970) Clin. Chim. Acta 30, 645.