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Study on the uterine mucosubstances accompanying the infertility effect of an intrauterine device
CLINICA
CCA
CHIMICA
ACTA
29
5268
STUDY
ON THE
INFERTILITY
TETSURO
ABE*,
UTERINE
EFFECT
** MASAHIKO
Departments of Obstetrics Medicine, Sendai (Japan) (Received
MUCOSUBSTANCES
ACCOMPANYING
OF AN INTRAUTERINE
ENDO*
and Gynecology*
*, AND ZENSAKU
THE
DEVICE?
YOSIZAWA*
and of Biochemistry**,
Tohoku
*
University
School
of
June 26, 1972)
SUMMARY
Endometrial mucosubstances (glycoproteins and mucopolysaccharides) have been investigated accompanying the infertility effect of an intrauterine device (IUD) under hormonal treatment. (I) The relative amounts of the total endometrial mucosubstances were increased with estrogen, but decreased with progesterone. Furthermore, higher values were observed in the IUD horns than in the h on-IUD horns in the hormone-treated animals. (2) Ion-exchange column chromatography on Dowex-r of the endometrial mucosubstances, expressed in terms of hexosamine, showed a remarkable alteration in the major NaCl fractions, 0.25 M, 0.5 RI and 0.75 M. The relative amounts of hexosamine in the 0.5 M and 0.75 M NaCl fractions from the IUD horns were higher than those from the non-IUD horns. (3) Electrophoretograms of the 0.5 M and 0.75 M NaCl fractions indicated the presence of three components: glycoprotein, sulfated glycoprotein and hyaluronic acid. (4) Histochemical study of the endometria of the rabbits showed the presence of PAS-positive substances in the lumina, the surface epithelia, the blood vessel wall and stroma, particularly in the lumina and the surface epithelia of the IUD horns. Moreover, the most intense staining was observed in the estrogen-treated animals. Thesubstances in the endometrial surface epithelia and the lumina in the estrogentreated animals also gave a positive reaction to alcian blue staining. (5) The results of the biochemical and histochemical studies suggest that the biosynthesis of endometrial mucosubstances, particularly glycoprotein and sulfated glycoprotein, was stimulated by estrogen, but this stimulation was prevented by progesterone. The results also suggest that the IUD accelerates estrogen stimulation, but suppresses the progesteronic effect. The present observations suggest that the IUD might change the metabolic pattern of endometrial mucosubstances. Therefore, the metabolic change of the endometrial mucosubstances, particularly of glycoprotein and sulfated glycoprotein, might play an important role in the infertility effect of the IUD.
t This work has been supported
by grants from the Population
Council,
New York,
Clin. Chim.
Acta,
N.Y.,
42
(U.S.A).
(1972) 29-35
30
ABE
et a/.
INTRODLTCTIOS
Although
an intrauterine
device
(IUD)
is known
to be a safe, reasonably
success-
ful and reversible method of fertility control and is widely used these days’, the mechanism of its action has not yet been elucidated. It has been suggested that the IUD might exert its principal action in the uterus by interfering with the implantation and dev,elopment of the fertilized ovum. On the other hand, endometrial mucosubstances are regarded as an indispensable factor providing a suitable environment for implantation of the fertilized ovum under hormonal control. In order to elucidate the mechanism of the contraceptive action of the IUD, its effect
on the endometrial
mucosubstances
of the rabbit
has been
studied.
EXPERIMENTAL
Material Pronase P was purchased from Kaken Kagaku Co. Ltd. Tokyo. An aqueous estradiol benzoate suspension (0.2 mg in I ml) and a progesterone solution (5 mg in I ml oil) were the products of the Sanzen Seiyaku Co. Ltd., Tokyo and supplied by the Yamanouchi Seiyaku Co. Ltd., Tokyo. Hyaluronic acid was prepared from human umbilical cord according to the method of Danishefsky and Bella2. Chondroitin sulfate A was supplied by the Seikagaku Kogyo Co. Ltd., Tokyo. Cellulose acetate membrane, Separax, was purchased materials were from a commercial Animals Adult female were ovariectomized.
from the source.
Joko
Sangyo
CO. Ltd.,
Tokyo.
Other
rabbits, about I year old and weighing approximately 2.5 to 3 kg, An IUD, a surgical silk suture of approximately 3 cm in length,
was inserted directly from the upper portion of the uterine cervix to the uterotubal junction of one of the two horns of the animals under aseptic conditions. This horn was designated as an IUD horn, while another one, without the IUD, a non-IUD horn. The rabbits were then fed ad libitunz for one month prior to hormonal treatments. The animals then received estradioi benzoate (0.1 mg per each animal, daily) intramuscularly for 3 days, providing the estrogen-treated rabbits. To obtain the estrogen-progesterone-treated rabbits, the estrogen-treated animals were injected intramuscularly with progesterone (5 mg per each animal, daily) for 3 days. Control animals were also examined without hormonal treatment. Each animal was sacrificed 24 h after the final injection
of hormone.
Separation of nmcosubstances from the rabbit endowm%m Each uterus taken from a sacrificed rabbit was washed with saline, andendoThe endometrial scrapings were metrium obtained by scraping from the uterus. sushomogenized with SO”,; ethanol and then heated at 100' for 3 min. The ethanolic pension was cooled in ice-water and subsequently centrifuged. The sediment thus obtained was washed successively with absolute ethanol and ether and then dried over CaCl, i?z rlaCZ~0.The dried powder was suspended in a 3-fold volume of distilled water, and the pH of the suspension was maintained at 8.0 with I il1NaOH during the incubation. To this suspension were added CaCl, (final concentration, 0.01 M) and Clin.
Chim.
Acta,
42
(1972)
29-35
MUCOSUBSTANCES
ACCO>fPANYINGTHE
INFERTILITY
31
EFFECT
pronase P (I mg/ro mg of the dried powder), and a few drops of toluene to prevent bacterial growth3. The mixture was incubated for 4 days at 37, the same amounts of pronase being added every day. The incubation was terminated by addition of 50% trichloroacetic acid to give a final concentration of 796 and the resulting precipitate was removed by centrifugation. The supernatant was dialysed against distilled water at room temperature. The non-dialysable fraction was concentrated to a small volume. To the concentrate was added ethanol to give a final concentration of Sot/,. The precipitate formed was collected by centrifugation, dissolved in distilled water, and reprecipitated with ethanol. The precipitate was collected and washed with ethanol and ether, and then dried over CaCl, in 7muo, yielding the endometrial mucosubstances. Fractionation
o_f the endometrial
mucosuOstances
An aqueous solution of the endometrial mucosubstances (1.5 N 4.5 mg in z ml) was applied to a column (I x 15 cm) of Dowex-r Sz, 200-400 mesh, chloride form, which was subsequently washed with IOO ml of water. Stepwise elution was carried out with 5o-ml batches each of NaC.1 solutions of increasing molarity from 0.25 hl to 5.0 M, and then with 50 ml of 4 IL1HCl, as shown in Fig. I. The eluant from each step Control
Estrogen-treated
-
Estrogen-progesterone-treated
1000
hexose
0 I I 0
hexasamine
non-No-horn w-horn non-w-horn
q w-horn
Fig. I. Elution diagrams obtained by Dowex-I column chromatography of the endomctrial mucosubstances. An aqueous solution of the crude mucosubstances (1.5 N 4.5 mg in 2 ml) was apphed to a column (I x 15 cm) of Dowex-1 x 2, zoo-400 mesh, chloride form, which was subscquently washed with water. Stepwise elution was carried out with KaCl and HCl as described in the text.
was dialysed against distilled water. The non-dialysable fraction was concentrated to dryness under reduced pressure. The dried residue was dissolved in I ml of water. The content of hexose in the water effluent, and of hexosamine in the other eluants were determined by the procedures of Dubois et a1.4 and Svennerholm”, respectively. Electrojhoresis
Electrophoresis was carried out on cellulose acetate membrane, Separax (6 x 22 cm) in formic acid-pyridine buffer (pH 3.0)~ at 0.5 m_4/cm for 50 min’. The substances were visualized by staining with toluidine blue and by the periodic acid-Schiff (PAS) reaction. Clin.
Chim.
Acta,
42
(1972) 29-35
ABE et al.
32 Histochemical examination The uteri of the experimental
animals were sectioned
at the midportion
of each
llorn. The specimens were fixed in Carnoy’s solution for 4 11, and then embedded in paraffin. Sections (6~ thickness) were stained with hematoxylin-eosine, toluidine blue, alcian blue and by the PAS reaction,
and examined
under the microscopes~3.
RESULTS
The weights of the endonletrial
scrapings
obtained
from the IITD h,>rns and the
non-IUD horns of control, estrogen-treated and estrogen-progesterone-treated rabbits are shown in Table 1. The data show that the hormonal treatments resulted in a TABLE
I
U-EIGHTSOF
THE
ENDOMETRIAL
SCRAPIKGS
Weights are expressed as average dry weight per rabbit. Estrogen-tveakdb
COtZtYOl~
Estrogen-pvogesterow-trmtedC
-
--Xon-I lJD Non-l UD .-._-_____I CD Wet weight (mg) 36.2 58.5 200.0 Dry weight (mg) 5-O 6.8 25.6 Numbers of animals used were: a, 13; b and C, 4.
~-
_..-.-_
Non-l UD -.-~-572.5 83.3
IUD 162.5 22.2
IUD 560.0 68.X
remarkable growth of endometrial tissues as noticed previously10911. The weight of the endometrial scrapings from the IUD horns was greater than that of the non-IUD horns in the control, whereas the reverse relation was observed under hormonal treatments. The relative amounts (pg/mg dry endometrial scraping) of the total endometrial mucosubstances obtained from the IUD horns and the non-IUD horns of control, estrogen-treated TABLE
and estrogen-progesterone-treated
rabbits
are shown in Table
II.
II
RELATIVE AMOUlirs
OF TOTAL
ENDOMETRIAL
MUCOSUBSTANCES
Relative amounts are expressed as ,uglmg dry endometrial scraping
Non-IUD IUD
CC7PdYOL
Estrogentreated
Estrogen-progesteronetreated
26. I
27.3 30.5
Ij.6
18.2
12.9
The values increased in the estrogen-treated animals, but decreased in the estrogenprogesterone-treated animals. Moreover, the values decreased in the IUD horns of control animals, but increased in the IUD horns of the hormone-treated animals compared to the non-IUD horns. Hution patterns obtained by ion-exchange column chromatography of the endometrial mucosubstances are shown in Fig. I. Remarkable alteration of the elution patterns was observed in the 0.25 M, 0.5 M and 0.75 M NaCl fractions. The relative amounts of hexosamine in the 0.5 M and 0.75 M fractions from the IUD horns were higher than those in the non-IUD horns. The difference in the values between the two Clin. Chim. Acta, 42 (x972) 29-35
MUCOSUBSTANCES
horns
ACCOMPANYING
was more pronounced
THE
INFERTILITY
in the estrogen-treated
animals. In the estrogen-progesterone-treated all the fractions except the H,O fractions
33
EFFECT
animals
than
in the control
animals, the amounts of hexosamine in were depressed in both horns, but the de-
pression was greater in the non-IUD horns than in the IUD horns. Electrophoresis was carried out on cellulose acetate membranes of the major NaCl eluants, namely the 0.25 11, 0.5 11 and 0.75 RI ?;aCl fractions, obtained by ionexchange column chromatography of the endomttrial mucosubstances. Since the electrophoretograms of the corresponding fractions from all the cases showed similar profiles, a representative one is shown in Fig. 2. As can be seen in the figure, the 0.25 M
-_-----;;: Gly 025 05 075 025 05 015 HA Chs-A MM
M
M
M
M
0
Fig. 2. Tracing of an electrophoretogram on cellulose acetate membrane of the 0.25 M, 0.5 M and 0.75 M NaCl fractions obtained by ion-exchange column chromatography of the endometrial mucosubstances from the non-IUD horns of the cstrogcn-treated rabbits. Electrophoresis was carried out in formic acid-pyridine buffer (pH 3.0) at 0.5 mh/cm for 50 min; staining was by PAS reaction (A) and toluidine blue (R). Gly, glycogen; HA, hyaluronic acid; Chs-A, chondroitin sulfate A.
fraction gave one broad band reacting positively to PAS staining, which migrated similarly to glycogen and glycoprotein. However, the 0.5 M fraction separated into two bands. One component stained positively with toluidine blue, and showed the same mobility as hyaluronic acid. The 0.75 Nl fraction also gave two bands reacting positively to PAS staining, but only one of them stained metachromatically with toluidine blue. This latter component migrated between hyaluronic acid and chondroitin sulfate A and showed a similar mobility to sulfated glycoproteins as reported previously12113. The PAS-positive components of the 0.5 M and 0.75 ill fractions migrated between glycogen and hyaluronic acid, and had similar mobilities to those of glycoproteins as reported previously12. Histochemical studies showed that the PAS-positive substances appeared in the lumina, the epithelia, the blood vessel wall and the stroma in the IUD horns as well in the non-IUD horns and in the surface epithelia in contact with the IUD. The colour of the staining was most intense in the estrogen-treated animals, intense in the estrogen-progesterone-treated animals and moderate in the control animals. The substances in the endometrial surface epithelia and lumina in the estrogen-treated animals also gave a positive reaction to alcian blue staining. DISCUSSION
metrial
The results of the present study show that the relative amounts of total endcmucosubstances in rabbits were increased by estrogen treatment, but deClin.
Chiwz.
Acta,
42 (1972)
29-35
ABE
34
et a/.
creased by progesterone treatment. Higher values were observed in the IUD horns than in the non-IUD horns under hormonal treatment. These findings indicate that the bios~ntllesis of endometrial nluc~)substances was stimulated by estrogen, but this stimulation was prevented by progesterone as noticed previouslylc~ll. Noreover, the present results suggest that the 11JD enhanced the estrogen stimulation, but suppressed the progesterone effect Ion-exchange column chromatography on Dowex-I of the cndometrial mucosubstances, expressed in terms of hexosamine, showed a remarkable alteration in the three major fractions, 0.25 >I, 0.5 %I and 0.75 it1 SaCI. The relative amounts of hexosamine in the 0.5 ,I1 and 0.75 JI NaCl fractions from the IUD horns were higher than those from the non-ICD horns. Since hexosamine is a representative constituent of mucosubstances, the values should indicate the contents of mucosubstanccs. Klectrophoretograms on cellulose acetate membrane of the 0.25 M, 0.5 M and 0.75 RI fractions showed the presence of three classes of Inucosubstance, glycoprotein, sulfated glycoprotein and hyaluronic acid. The biosynthesis of these components seems to be stimulated remarkably by estrogen, but suppressed by progesterone. Some of these components might be responsible for the enhancement of the estrogen effect by the IUD. f-[istochemical study of the endometria of the rabbit uterine horns revealed the presence of PAS-positive substances in the lumina, the surface epithelia, the blood vessel wall and the stroma. This was most marked in the lumina and the surface epithelia of the IUD horns and in the surface epithelia in contact with the IUD. Staining was most intense in the estrogen-treated animals. The substances in the endometrial surface epithelia and the lumina in the estrogen-treated animals also gave a positive reaction to alcian blue staining. The results of the bioc~~emical and histochemical studies thus far obtained suggest that the IUD accelerates the estrogen stimulation of the biosynthesis of endometrial mucosubstances such as glycoprotein and sulfated glycoprotein, but suppresses the progesterone effect, giving abnormal amounts of these components in the endometrium, particularly in the lumina and the surface epithelia under the hormonal treatment. Several authors have reported the effect of the IUD on the Inetabolism of endometrial mucosubstances. Based on the results of histological study, Hall et aE.lP recorded the failure of depolymerization of mucopolysaccharides in the ground substance of the endometris of IUD-wearing women. On the other hand, Cooper et a1.15 reported that IUD depressed the hexosamine concentration in the endometria of rabbits, but the hexosarnine and uranic acid concentrations in the endometrium were nearly doubled in the IUD areas of the uterine horns of the cowl”. In case of ewes, higher hexosamine concentrations were observed in the IUD horns than in the nonIUD horns of the same ewes 17. In addition, the results of the present study show that the IUD increased the relative contents of hexosamine of the rabbit endometria. These observations indicate that the effect of the IUD on the endometrial mucosubstances seem to depend on the species and the hormonal milieu of the esperimental conditions. ‘rakakuwa18,19 studied the endometrial mucosubstances of rabbits and humans with IUD, and suggested a depression of the glycogen concentration in the rabbit endometria and a shift of the normal metabolic pattern of human endometrial mucoC&n. Chillz. ncta,
42 (1972) 29-35
MCCOSLJUSTANCES
ACCOMPASYING
THE ISFERTILITY
EFFECT
35
polysaccharides in the presence of the IUD under endogenous hormonal control. These changes might indicate an unsuitable endometrial environment for implantation of the fertilized ovum. The metabolic shift suggested by Takakuwal*~ls was similar to that found in the present study on rabbits. This change in the endometrial mucosubstances probably plays an important role in the infertility effect of the IUD. KEFERENCES I 2 3 4
I’oInlation Council, Stz~dzr~zn Fan&y Plaxning, So. 27 (1968). L. UASISHEFSKY ASU h. BELLA, JR., ,I, Riol. Chrm., 241 (1966) 143. II; (1966) 257. S. INOUE Aln Z. yOSIZAW.4, Arch. Biochem. Bzophys., >I. DUBOIS, Ei. .\. GILLES, J. li. HAMILTON, P. .A. RERHRS ASD F. SMITH, .4nal. Chem.,
28
5 6 7 8 9 IO II 12 13
(1956) 350. L. SVESXERHOLM, Acta Sot. ~Ucd. C’psaliensis, 61 (19j6) 287. 52. B. MATHEWS, B&him. Hiophys. Acta, 48 (1961) 402. A. KIMURA AKD K. TSURUMI, ,I. Biochem., 65 (1969) 303. D. HAMERMaK, .‘ciewcr, 122 (19j5) 924. B. 1
14 15 16 17 I8 rg
(1970) 4’9. H. H. HALL, il. SEDLIS, I. CHABO~ AND M. L. STOSE, Amev. J. Obstet. Gynecol., 33. S. COOPER, G. 1. GREEX ASD H. W. HAWK, J. Animal Sci., 30 (1970) 767. B. S. COOPER AND H. \\‘. HAWK, Fertilzty Stevility, 19 (1968) 310. B. S. COOPER ASD H. IV. HAWK, J, Animal Sci., 27 (1966) 1373. .A..TAKAKUWA, .[apakl. J. Obstet. Gynecol.. 20 (1968) 365. _I. T.~KAKWVA, Japan. J. Obstet. Gynccol., LO (1968) 486. Clin. Chim.
Acta,
93 (1965)
42 (1972)
1031.
29-35
CCA
CHIMICA
ACTA
29
5268
STUDY
ON THE
INFERTILITY
TETSURO
ABE*,
UTERINE
EFFECT
** MASAHIKO
Departments of Obstetrics Medicine, Sendai (Japan) (Received
MUCOSUBSTANCES
ACCOMPANYING
OF AN INTRAUTERINE
ENDO*
and Gynecology*
*, AND ZENSAKU
THE
DEVICE?
YOSIZAWA*
and of Biochemistry**,
Tohoku
*
University
School
of
June 26, 1972)
SUMMARY
Endometrial mucosubstances (glycoproteins and mucopolysaccharides) have been investigated accompanying the infertility effect of an intrauterine device (IUD) under hormonal treatment. (I) The relative amounts of the total endometrial mucosubstances were increased with estrogen, but decreased with progesterone. Furthermore, higher values were observed in the IUD horns than in the h on-IUD horns in the hormone-treated animals. (2) Ion-exchange column chromatography on Dowex-r of the endometrial mucosubstances, expressed in terms of hexosamine, showed a remarkable alteration in the major NaCl fractions, 0.25 M, 0.5 RI and 0.75 M. The relative amounts of hexosamine in the 0.5 M and 0.75 M NaCl fractions from the IUD horns were higher than those from the non-IUD horns. (3) Electrophoretograms of the 0.5 M and 0.75 M NaCl fractions indicated the presence of three components: glycoprotein, sulfated glycoprotein and hyaluronic acid. (4) Histochemical study of the endometria of the rabbits showed the presence of PAS-positive substances in the lumina, the surface epithelia, the blood vessel wall and stroma, particularly in the lumina and the surface epithelia of the IUD horns. Moreover, the most intense staining was observed in the estrogen-treated animals. Thesubstances in the endometrial surface epithelia and the lumina in the estrogentreated animals also gave a positive reaction to alcian blue staining. (5) The results of the biochemical and histochemical studies suggest that the biosynthesis of endometrial mucosubstances, particularly glycoprotein and sulfated glycoprotein, was stimulated by estrogen, but this stimulation was prevented by progesterone. The results also suggest that the IUD accelerates estrogen stimulation, but suppresses the progesteronic effect. The present observations suggest that the IUD might change the metabolic pattern of endometrial mucosubstances. Therefore, the metabolic change of the endometrial mucosubstances, particularly of glycoprotein and sulfated glycoprotein, might play an important role in the infertility effect of the IUD.
t This work has been supported
by grants from the Population
Council,
New York,
Clin. Chim.
Acta,
N.Y.,
42
(U.S.A).
(1972) 29-35
30
ABE
et a/.
INTRODLTCTIOS
Although
an intrauterine
device
(IUD)
is known
to be a safe, reasonably
success-
ful and reversible method of fertility control and is widely used these days’, the mechanism of its action has not yet been elucidated. It has been suggested that the IUD might exert its principal action in the uterus by interfering with the implantation and dev,elopment of the fertilized ovum. On the other hand, endometrial mucosubstances are regarded as an indispensable factor providing a suitable environment for implantation of the fertilized ovum under hormonal control. In order to elucidate the mechanism of the contraceptive action of the IUD, its effect
on the endometrial
mucosubstances
of the rabbit
has been
studied.
EXPERIMENTAL
Material Pronase P was purchased from Kaken Kagaku Co. Ltd. Tokyo. An aqueous estradiol benzoate suspension (0.2 mg in I ml) and a progesterone solution (5 mg in I ml oil) were the products of the Sanzen Seiyaku Co. Ltd., Tokyo and supplied by the Yamanouchi Seiyaku Co. Ltd., Tokyo. Hyaluronic acid was prepared from human umbilical cord according to the method of Danishefsky and Bella2. Chondroitin sulfate A was supplied by the Seikagaku Kogyo Co. Ltd., Tokyo. Cellulose acetate membrane, Separax, was purchased materials were from a commercial Animals Adult female were ovariectomized.
from the source.
Joko
Sangyo
CO. Ltd.,
Tokyo.
Other
rabbits, about I year old and weighing approximately 2.5 to 3 kg, An IUD, a surgical silk suture of approximately 3 cm in length,
was inserted directly from the upper portion of the uterine cervix to the uterotubal junction of one of the two horns of the animals under aseptic conditions. This horn was designated as an IUD horn, while another one, without the IUD, a non-IUD horn. The rabbits were then fed ad libitunz for one month prior to hormonal treatments. The animals then received estradioi benzoate (0.1 mg per each animal, daily) intramuscularly for 3 days, providing the estrogen-treated rabbits. To obtain the estrogen-progesterone-treated rabbits, the estrogen-treated animals were injected intramuscularly with progesterone (5 mg per each animal, daily) for 3 days. Control animals were also examined without hormonal treatment. Each animal was sacrificed 24 h after the final injection
of hormone.
Separation of nmcosubstances from the rabbit endowm%m Each uterus taken from a sacrificed rabbit was washed with saline, andendoThe endometrial scrapings were metrium obtained by scraping from the uterus. sushomogenized with SO”,; ethanol and then heated at 100' for 3 min. The ethanolic pension was cooled in ice-water and subsequently centrifuged. The sediment thus obtained was washed successively with absolute ethanol and ether and then dried over CaCl, i?z rlaCZ~0.The dried powder was suspended in a 3-fold volume of distilled water, and the pH of the suspension was maintained at 8.0 with I il1NaOH during the incubation. To this suspension were added CaCl, (final concentration, 0.01 M) and Clin.
Chim.
Acta,
42
(1972)
29-35
MUCOSUBSTANCES
ACCO>fPANYINGTHE
INFERTILITY
31
EFFECT
pronase P (I mg/ro mg of the dried powder), and a few drops of toluene to prevent bacterial growth3. The mixture was incubated for 4 days at 37, the same amounts of pronase being added every day. The incubation was terminated by addition of 50% trichloroacetic acid to give a final concentration of 796 and the resulting precipitate was removed by centrifugation. The supernatant was dialysed against distilled water at room temperature. The non-dialysable fraction was concentrated to a small volume. To the concentrate was added ethanol to give a final concentration of Sot/,. The precipitate formed was collected by centrifugation, dissolved in distilled water, and reprecipitated with ethanol. The precipitate was collected and washed with ethanol and ether, and then dried over CaCl, in 7muo, yielding the endometrial mucosubstances. Fractionation
o_f the endometrial
mucosuOstances
An aqueous solution of the endometrial mucosubstances (1.5 N 4.5 mg in z ml) was applied to a column (I x 15 cm) of Dowex-r Sz, 200-400 mesh, chloride form, which was subsequently washed with IOO ml of water. Stepwise elution was carried out with 5o-ml batches each of NaC.1 solutions of increasing molarity from 0.25 hl to 5.0 M, and then with 50 ml of 4 IL1HCl, as shown in Fig. I. The eluant from each step Control
Estrogen-treated
-
Estrogen-progesterone-treated
1000
hexose
0 I I 0
hexasamine
non-No-horn w-horn non-w-horn
q w-horn
Fig. I. Elution diagrams obtained by Dowex-I column chromatography of the endomctrial mucosubstances. An aqueous solution of the crude mucosubstances (1.5 N 4.5 mg in 2 ml) was apphed to a column (I x 15 cm) of Dowex-1 x 2, zoo-400 mesh, chloride form, which was subscquently washed with water. Stepwise elution was carried out with KaCl and HCl as described in the text.
was dialysed against distilled water. The non-dialysable fraction was concentrated to dryness under reduced pressure. The dried residue was dissolved in I ml of water. The content of hexose in the water effluent, and of hexosamine in the other eluants were determined by the procedures of Dubois et a1.4 and Svennerholm”, respectively. Electrojhoresis
Electrophoresis was carried out on cellulose acetate membrane, Separax (6 x 22 cm) in formic acid-pyridine buffer (pH 3.0)~ at 0.5 m_4/cm for 50 min’. The substances were visualized by staining with toluidine blue and by the periodic acid-Schiff (PAS) reaction. Clin.
Chim.
Acta,
42
(1972) 29-35
ABE et al.
32 Histochemical examination The uteri of the experimental
animals were sectioned
at the midportion
of each
llorn. The specimens were fixed in Carnoy’s solution for 4 11, and then embedded in paraffin. Sections (6~ thickness) were stained with hematoxylin-eosine, toluidine blue, alcian blue and by the PAS reaction,
and examined
under the microscopes~3.
RESULTS
The weights of the endonletrial
scrapings
obtained
from the IITD h,>rns and the
non-IUD horns of control, estrogen-treated and estrogen-progesterone-treated rabbits are shown in Table 1. The data show that the hormonal treatments resulted in a TABLE
I
U-EIGHTSOF
THE
ENDOMETRIAL
SCRAPIKGS
Weights are expressed as average dry weight per rabbit. Estrogen-tveakdb
COtZtYOl~
Estrogen-pvogesterow-trmtedC
-
--Xon-I lJD Non-l UD .-._-_____I CD Wet weight (mg) 36.2 58.5 200.0 Dry weight (mg) 5-O 6.8 25.6 Numbers of animals used were: a, 13; b and C, 4.
~-
_..-.-_
Non-l UD -.-~-572.5 83.3
IUD 162.5 22.2
IUD 560.0 68.X
remarkable growth of endometrial tissues as noticed previously10911. The weight of the endometrial scrapings from the IUD horns was greater than that of the non-IUD horns in the control, whereas the reverse relation was observed under hormonal treatments. The relative amounts (pg/mg dry endometrial scraping) of the total endometrial mucosubstances obtained from the IUD horns and the non-IUD horns of control, estrogen-treated TABLE
and estrogen-progesterone-treated
rabbits
are shown in Table
II.
II
RELATIVE AMOUlirs
OF TOTAL
ENDOMETRIAL
MUCOSUBSTANCES
Relative amounts are expressed as ,uglmg dry endometrial scraping
Non-IUD IUD
CC7PdYOL
Estrogentreated
Estrogen-progesteronetreated
26. I
27.3 30.5
Ij.6
18.2
12.9
The values increased in the estrogen-treated animals, but decreased in the estrogenprogesterone-treated animals. Moreover, the values decreased in the IUD horns of control animals, but increased in the IUD horns of the hormone-treated animals compared to the non-IUD horns. Hution patterns obtained by ion-exchange column chromatography of the endometrial mucosubstances are shown in Fig. I. Remarkable alteration of the elution patterns was observed in the 0.25 M, 0.5 M and 0.75 M NaCl fractions. The relative amounts of hexosamine in the 0.5 M and 0.75 M fractions from the IUD horns were higher than those in the non-IUD horns. The difference in the values between the two Clin. Chim. Acta, 42 (x972) 29-35
MUCOSUBSTANCES
horns
ACCOMPANYING
was more pronounced
THE
INFERTILITY
in the estrogen-treated
animals. In the estrogen-progesterone-treated all the fractions except the H,O fractions
33
EFFECT
animals
than
in the control
animals, the amounts of hexosamine in were depressed in both horns, but the de-
pression was greater in the non-IUD horns than in the IUD horns. Electrophoresis was carried out on cellulose acetate membranes of the major NaCl eluants, namely the 0.25 11, 0.5 11 and 0.75 RI ?;aCl fractions, obtained by ionexchange column chromatography of the endomttrial mucosubstances. Since the electrophoretograms of the corresponding fractions from all the cases showed similar profiles, a representative one is shown in Fig. 2. As can be seen in the figure, the 0.25 M
-_-----;;: Gly 025 05 075 025 05 015 HA Chs-A MM
M
M
M
M
0
Fig. 2. Tracing of an electrophoretogram on cellulose acetate membrane of the 0.25 M, 0.5 M and 0.75 M NaCl fractions obtained by ion-exchange column chromatography of the endometrial mucosubstances from the non-IUD horns of the cstrogcn-treated rabbits. Electrophoresis was carried out in formic acid-pyridine buffer (pH 3.0) at 0.5 mh/cm for 50 min; staining was by PAS reaction (A) and toluidine blue (R). Gly, glycogen; HA, hyaluronic acid; Chs-A, chondroitin sulfate A.
fraction gave one broad band reacting positively to PAS staining, which migrated similarly to glycogen and glycoprotein. However, the 0.5 M fraction separated into two bands. One component stained positively with toluidine blue, and showed the same mobility as hyaluronic acid. The 0.75 Nl fraction also gave two bands reacting positively to PAS staining, but only one of them stained metachromatically with toluidine blue. This latter component migrated between hyaluronic acid and chondroitin sulfate A and showed a similar mobility to sulfated glycoproteins as reported previously12113. The PAS-positive components of the 0.5 M and 0.75 ill fractions migrated between glycogen and hyaluronic acid, and had similar mobilities to those of glycoproteins as reported previously12. Histochemical studies showed that the PAS-positive substances appeared in the lumina, the epithelia, the blood vessel wall and the stroma in the IUD horns as well in the non-IUD horns and in the surface epithelia in contact with the IUD. The colour of the staining was most intense in the estrogen-treated animals, intense in the estrogen-progesterone-treated animals and moderate in the control animals. The substances in the endometrial surface epithelia and lumina in the estrogen-treated animals also gave a positive reaction to alcian blue staining. DISCUSSION
metrial
The results of the present study show that the relative amounts of total endcmucosubstances in rabbits were increased by estrogen treatment, but deClin.
Chiwz.
Acta,
42 (1972)
29-35
ABE
34
et a/.
creased by progesterone treatment. Higher values were observed in the IUD horns than in the non-IUD horns under hormonal treatment. These findings indicate that the bios~ntllesis of endometrial nluc~)substances was stimulated by estrogen, but this stimulation was prevented by progesterone as noticed previouslylc~ll. Noreover, the present results suggest that the 11JD enhanced the estrogen stimulation, but suppressed the progesterone effect Ion-exchange column chromatography on Dowex-I of the cndometrial mucosubstances, expressed in terms of hexosamine, showed a remarkable alteration in the three major fractions, 0.25 >I, 0.5 %I and 0.75 it1 SaCI. The relative amounts of hexosamine in the 0.5 ,I1 and 0.75 JI NaCl fractions from the IUD horns were higher than those from the non-ICD horns. Since hexosamine is a representative constituent of mucosubstances, the values should indicate the contents of mucosubstanccs. Klectrophoretograms on cellulose acetate membrane of the 0.25 M, 0.5 M and 0.75 RI fractions showed the presence of three classes of Inucosubstance, glycoprotein, sulfated glycoprotein and hyaluronic acid. The biosynthesis of these components seems to be stimulated remarkably by estrogen, but suppressed by progesterone. Some of these components might be responsible for the enhancement of the estrogen effect by the IUD. f-[istochemical study of the endometria of the rabbit uterine horns revealed the presence of PAS-positive substances in the lumina, the surface epithelia, the blood vessel wall and the stroma. This was most marked in the lumina and the surface epithelia of the IUD horns and in the surface epithelia in contact with the IUD. Staining was most intense in the estrogen-treated animals. The substances in the endometrial surface epithelia and the lumina in the estrogen-treated animals also gave a positive reaction to alcian blue staining. The results of the bioc~~emical and histochemical studies thus far obtained suggest that the IUD accelerates the estrogen stimulation of the biosynthesis of endometrial mucosubstances such as glycoprotein and sulfated glycoprotein, but suppresses the progesterone effect, giving abnormal amounts of these components in the endometrium, particularly in the lumina and the surface epithelia under the hormonal treatment. Several authors have reported the effect of the IUD on the Inetabolism of endometrial mucosubstances. Based on the results of histological study, Hall et aE.lP recorded the failure of depolymerization of mucopolysaccharides in the ground substance of the endometris of IUD-wearing women. On the other hand, Cooper et a1.15 reported that IUD depressed the hexosamine concentration in the endometria of rabbits, but the hexosarnine and uranic acid concentrations in the endometrium were nearly doubled in the IUD areas of the uterine horns of the cowl”. In case of ewes, higher hexosamine concentrations were observed in the IUD horns than in the nonIUD horns of the same ewes 17. In addition, the results of the present study show that the IUD increased the relative contents of hexosamine of the rabbit endometria. These observations indicate that the effect of the IUD on the endometrial mucosubstances seem to depend on the species and the hormonal milieu of the esperimental conditions. ‘rakakuwa18,19 studied the endometrial mucosubstances of rabbits and humans with IUD, and suggested a depression of the glycogen concentration in the rabbit endometria and a shift of the normal metabolic pattern of human endometrial mucoC&n. Chillz. ncta,
42 (1972) 29-35
MCCOSLJUSTANCES
ACCOMPASYING
THE ISFERTILITY
EFFECT
35
polysaccharides in the presence of the IUD under endogenous hormonal control. These changes might indicate an unsuitable endometrial environment for implantation of the fertilized ovum. The metabolic shift suggested by Takakuwal*~ls was similar to that found in the present study on rabbits. This change in the endometrial mucosubstances probably plays an important role in the infertility effect of the IUD. KEFERENCES I 2 3 4
I’oInlation Council, Stz~dzr~zn Fan&y Plaxning, So. 27 (1968). L. UASISHEFSKY ASU h. BELLA, JR., ,I, Riol. Chrm., 241 (1966) 143. II; (1966) 257. S. INOUE Aln Z. yOSIZAW.4, Arch. Biochem. Bzophys., >I. DUBOIS, Ei. .\. GILLES, J. li. HAMILTON, P. .A. RERHRS ASD F. SMITH, .4nal. Chem.,
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(1956) 350. L. SVESXERHOLM, Acta Sot. ~Ucd. C’psaliensis, 61 (19j6) 287. 52. B. MATHEWS, B&him. Hiophys. Acta, 48 (1961) 402. A. KIMURA AKD K. TSURUMI, ,I. Biochem., 65 (1969) 303. D. HAMERMaK, .‘ciewcr, 122 (19j5) 924. B. 1
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(1970) 4’9. H. H. HALL, il. SEDLIS, I. CHABO~ AND M. L. STOSE, Amev. J. Obstet. Gynecol., 33. S. COOPER, G. 1. GREEX ASD H. W. HAWK, J. Animal Sci., 30 (1970) 767. B. S. COOPER AND H. \\‘. HAWK, Fertilzty Stevility, 19 (1968) 310. B. S. COOPER ASD H. IV. HAWK, J, Animal Sci., 27 (1966) 1373. .A..TAKAKUWA, .[apakl. J. Obstet. Gynecol.. 20 (1968) 365. _I. T.~KAKWVA, Japan. J. Obstet. Gynccol., LO (1968) 486. Clin. Chim.
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