FERTILITY AND STERILITY Copyright © 1976 The American Fertility Society
Vo!' 27, No.3, March 1976 Printed in U.8A.
SOME PROPERTIES OF HUMAN ENDOMETRIAL ALKALINE PHOSPHATASE EARLE W. WILSON, M.B., CH.B., M.D., M.R.C.O.G.
Department of Obstetrics and Gynaecology, University of Otago Medical School, Dunedin, New Zealand
A study of some biochemical properties of alkaline phosphatase derived from human endometrium has been undertaken. The endometrial enzyme has been shown to be different from alkaline phosphatases obtained from placenta and small intestine. Endometrial alkaline phosphatase is inhibited by sodium deoxycholate but not by L-phenylalanine; it is completely inhibited by 3 M urea. Magnesium ions have no significant effect on the endometrial enzyme. No differences in biochemical properties were ob-
served when alkaline phosphatase from follicular phase endometrium was compared with that from luteal phase tissue. Acrylamide gel electrophoresis showed a single, constant band of enzyme at all stages of the cycle. It is concluded that, despite the cyclic appearance of alkaline phosphatase in endometrial glands and the constant presence of the enzyme in blood vessels, there is but one variety of alkaline phosphatase in human endometrium.
The presence of alkaline phosphatase in the human endometrium has often been described.l,a McKay et al.,4 in a comprehensive account of the activity of this enzyme in human endometrium throughout the cycle, described enzyme activity reaching a maximum at different sites in the endometrium at different stages of the cycle. During the follicular phase and in the early part of the luteal phase there is pronounced enzyme activity in endometrial gland cells, and alkaline phosphatase appears in the uterine secretion. After about day 20 there is a marked decline. in glandular alkaline phosphatase, and by day 23 or day 24 there is little or no enzyme activity in endometrial glands. Throughout the whole of the cycle there is alkaline phosphatase activity
in the endothelium of arterioles in the endometrium. Because of the great increase in the blood supply to the endometrium during the luteal phase there is an apparent increase in the amount of enzyme activity at this site during the second half of the luteal phase. Because the enzyme appears at different sites within the endometrium, and because there are different levels of activity at these sites at any given time during the cycle, the possibility exists that the enzyme may itself be different in these separate situations. Although the varieties of alkaline phosphatase in placenta, intestine, liver, bone, and kidney have been characterized biochemically,5. 6 there has been no published description of the catalytic properties of human endometrial alkaline phosphatase. This report describes attempts to separate isoenzymes of alkaline phospha-
Received March 25, 1975.
299
WILSON
300
tase in human endometrium during different stages of the menstrual cycle, using polyacrylamide gel electrophoresis, and describes some of the catalytic properties of the endometrial enzyme. Human alkaline phosphatases are inhibited to different extents by urea, Lphenylalanine, and bile acids, according to the tissue of origin of the enzyme. Therefore, these substances have been investigated for their effects on endometrial alkaline phosphatase. Magnesium ions are known to stimulate phosphatase activity, and the effect of magnesium on endometrial alkaline phosphatase has also been studied. Because of the difficulties of the procedures with only small amounts of enzyme available and because of uncertainty about the meaning, or use, of the values,1 no attempt was made to estimate values for the Michaelis constant. MATERIALS AND METHODS
Endometrium was obtained by uterine curettage from women with apparently normal menstrual cycles who were being investigated as part of an infertility work-up where a male fault was known to exist, or who were subjected to curettage during a sterilization procedure. Specimens so obtained were divided into two portions, one of which was examined histologically to confirm that the tissue was normal for the cycle date, the criteria used being those described by Noyes et al. 8
March 1976
The remammg portion was either used fresh or stored frozen until used. Only endometrium that was histologically appropriate for the cycle date at which it was obtained was used in this study. Human small intestine was obtained at autopsy within 24 hours of death; the tissue was washed in water and freed of fat and connective tissue before use. Fresh human placenta was cut into l-cm cubes and washed free of blood before use. Tissue was homogenized with water and incubated with n-butyl alcohol for 30 minutes. The aqueous extracts were dialyzed against 20% polyvinyl pyrrolidone (MW 12,000) solution for 12 hours at 4° C. Twenty-four specimens of endometrium were used for electrophoretic studies. Six specimens were obtained between cycle days 8 and 16, ten specimens between days 17 and 22, and eight specimens on day 23 or later. Electrophoresis was carried out on 7% acrylamide gel, using a single buffer system of 0.0337 M Tris-borate at pH 9.5. The applied current was 2.5 mA/column at constant voltage. Phosphatase activity was demonstrated in gels by incubation for 2 to 4 hours in a solution of 50 mg of fast blue RR salt and 300 mg of sodium a-naphthyl-phosphate in 0.0337 M Tris-borate buffer at pH 9.5. Preliminary studies suggested that there was no difference in the inhibition characteristics of alkaline phosphatase from tissue obtained at various times of
TABLE 1. Effect of Magnesium Ions and L-Phenylalanine on Human Endometrial, Intestinal, and Placental Alkaline Phosphatase Activity Phenol liberated" from phenyl phosphate, pH 10.1
Tissue Control
Mg2+, 5 mM
L-Phenylalanine, 5 rnM
J.LMlminlliter
Endometriunf Follicular phase Luteal phase Intestine Placenta
62.9 38.1 49.7 181.0
± ± ± ±
4.17 3.11 3.11 8.2
67.2 38.6 59.3 191.7
Results are the means of five observations ± standard deviations. bEndometrium was pooled from three subjects for each cycle phase.
a
± ± ± ±
6.29 1.72 3.63 7.81
44.6 25.9 16.7 81.7
± ± ± ±
4.25 2.58 2.84 4.3
\ Vol. 27, No.3
ENDOMETRIAL ALKALINE PHOSPHATASE
the cycle. Because of the small amount of enzyme activity present in late luteal phase tissue it was necessary to use pooled specimens. Endometrium was obtained, as above, from six more women and was divided into two pools. One pool was derived from three women who were between cycle days 10 and 16 and the other from three women between cycle days 22 and 25. The results reported for the inhibition studies are derived from the enzyme extracts from these pools, termed follicular phase and luteal phase tissue, respectively. The rate of hydrolysis of phenyl phosphate in the presence and absence of magnesium ions as magnesium chloride, at a concentration of 5 mM, was studied with a reaction mixture of 5 mM phenyl phosphate and 0.01 M 4-aminoantipyrine in a sodium carbonate-bicarbonate buffet' at pH 10.1. This formed the basic reaction mixture for the remaining studies. The effects of sodium deoxycholate, L-phenylalanine, and increasing concentrations of urea on the hydrolysis of phenyl phosphate by alkaline phosphatase derived from intestine, placenta, and endometrium were also studied. The rate of hydrolysis in the presence of 5 mM sodium deoxycholate was compared with that in control solutions containing no sodium deoxycholate. A similar reaction mixture was used to study the effects of 5 mM L-phenylalanine. Control solutions contained no phenylalanine. The concentration of urea required
301
to produce irreversible inactivation of phosphatase activity in the three types of tissue was determined. Tissue extracts were diluted with equal volumes of urea solutions to give final urea concentrations of 2,3,4,5, 6, 7, and 8 M. The solutions were allowed to stand at room temperature for 2 hours, at the end of which time the rate of phenyl phosphate hydrolysis was determined for each solution. RESULTS
Electrophoretic studies showed a single, constant band of enzyme activity in all 24 specimens. In particular, no difference was discernible between the single band of alkaline phosphatase in endometrium from late in the luteal phase, i.e., after cycle day 22, and that in the follicular phase tissue up to day 16. the endometrial enzyme appeared in a position anodal to the intestinal enzyme and cathodal to the placental enzyme. The results of the inhibition studies showed that endometrial alkaline phosphatase was slightly inhibited by L-phenylalanine, but not to the extent that the intestinal or placental enzymes were inhibited by this agent (Table 1). Magnesium ions had no significant effect on the endometrial or placental enzymes, although the activity of both enzymes was increased slightly in the presence of magnesium. The intestinal enzyme was increased to a greater extent in the presence of magnesium.
TABLE 2. Effect of Sodium Deoxycholate on Human Endometrial, Intestinal, and Placental Alkaline Phosphatase Activity Phenol liberated' from phenyl phosphate, pH 9.9 Tissue
Control
Sodium deoxycholate, 5 mM
% Control
/LMlminlliter
Endometrium" Follicular phase Luteal phase Intestine Placenta a Results
57.6 37.4 56.9 295.5
± 5.82 ± 2.81 ±
4.67
± 12.03
29.9 16.1 55.6 303.0
± 4.3
2.10 5.6 ± 20.27
are the means of five observations ± standard deviation. bEndometrium was pooled from three subjects for each cycle phase.
± ±
51.8 43 98 102.5
March 1976
WILSON
302
Sodium deoxycholate inhibited the endometrial enzyme but not the placental or intestinal enzymes (Table 2). Urea produced complete inhibition of the endometrial enzyme at a concentration of 3 M (Table 3). The intestinal enzyme was similarly inhibited by 5 M urea, while the placental enzyme was unaffected by 8 M urea. There were no differences in the enzyme derived from the luteal and follicular phases of the cycle with regard to Lphenylalanine or bile acid inhibition or inactivation by urea. DISCUSSION
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Vo!' 27, No.3, March 1976 Printed in U.8A.
SOME PROPERTIES OF HUMAN ENDOMETRIAL ALKALINE PHOSPHATASE EARLE W. WILSON, M.B., CH.B., M.D., M.R.C.O.G.
Department of Obstetrics and Gynaecology, University of Otago Medical School, Dunedin, New Zealand
A study of some biochemical properties of alkaline phosphatase derived from human endometrium has been undertaken. The endometrial enzyme has been shown to be different from alkaline phosphatases obtained from placenta and small intestine. Endometrial alkaline phosphatase is inhibited by sodium deoxycholate but not by L-phenylalanine; it is completely inhibited by 3 M urea. Magnesium ions have no significant effect on the endometrial enzyme. No differences in biochemical properties were ob-
served when alkaline phosphatase from follicular phase endometrium was compared with that from luteal phase tissue. Acrylamide gel electrophoresis showed a single, constant band of enzyme at all stages of the cycle. It is concluded that, despite the cyclic appearance of alkaline phosphatase in endometrial glands and the constant presence of the enzyme in blood vessels, there is but one variety of alkaline phosphatase in human endometrium.
The presence of alkaline phosphatase in the human endometrium has often been described.l,a McKay et al.,4 in a comprehensive account of the activity of this enzyme in human endometrium throughout the cycle, described enzyme activity reaching a maximum at different sites in the endometrium at different stages of the cycle. During the follicular phase and in the early part of the luteal phase there is pronounced enzyme activity in endometrial gland cells, and alkaline phosphatase appears in the uterine secretion. After about day 20 there is a marked decline. in glandular alkaline phosphatase, and by day 23 or day 24 there is little or no enzyme activity in endometrial glands. Throughout the whole of the cycle there is alkaline phosphatase activity
in the endothelium of arterioles in the endometrium. Because of the great increase in the blood supply to the endometrium during the luteal phase there is an apparent increase in the amount of enzyme activity at this site during the second half of the luteal phase. Because the enzyme appears at different sites within the endometrium, and because there are different levels of activity at these sites at any given time during the cycle, the possibility exists that the enzyme may itself be different in these separate situations. Although the varieties of alkaline phosphatase in placenta, intestine, liver, bone, and kidney have been characterized biochemically,5. 6 there has been no published description of the catalytic properties of human endometrial alkaline phosphatase. This report describes attempts to separate isoenzymes of alkaline phospha-
Received March 25, 1975.
299
WILSON
300
tase in human endometrium during different stages of the menstrual cycle, using polyacrylamide gel electrophoresis, and describes some of the catalytic properties of the endometrial enzyme. Human alkaline phosphatases are inhibited to different extents by urea, Lphenylalanine, and bile acids, according to the tissue of origin of the enzyme. Therefore, these substances have been investigated for their effects on endometrial alkaline phosphatase. Magnesium ions are known to stimulate phosphatase activity, and the effect of magnesium on endometrial alkaline phosphatase has also been studied. Because of the difficulties of the procedures with only small amounts of enzyme available and because of uncertainty about the meaning, or use, of the values,1 no attempt was made to estimate values for the Michaelis constant. MATERIALS AND METHODS
Endometrium was obtained by uterine curettage from women with apparently normal menstrual cycles who were being investigated as part of an infertility work-up where a male fault was known to exist, or who were subjected to curettage during a sterilization procedure. Specimens so obtained were divided into two portions, one of which was examined histologically to confirm that the tissue was normal for the cycle date, the criteria used being those described by Noyes et al. 8
March 1976
The remammg portion was either used fresh or stored frozen until used. Only endometrium that was histologically appropriate for the cycle date at which it was obtained was used in this study. Human small intestine was obtained at autopsy within 24 hours of death; the tissue was washed in water and freed of fat and connective tissue before use. Fresh human placenta was cut into l-cm cubes and washed free of blood before use. Tissue was homogenized with water and incubated with n-butyl alcohol for 30 minutes. The aqueous extracts were dialyzed against 20% polyvinyl pyrrolidone (MW 12,000) solution for 12 hours at 4° C. Twenty-four specimens of endometrium were used for electrophoretic studies. Six specimens were obtained between cycle days 8 and 16, ten specimens between days 17 and 22, and eight specimens on day 23 or later. Electrophoresis was carried out on 7% acrylamide gel, using a single buffer system of 0.0337 M Tris-borate at pH 9.5. The applied current was 2.5 mA/column at constant voltage. Phosphatase activity was demonstrated in gels by incubation for 2 to 4 hours in a solution of 50 mg of fast blue RR salt and 300 mg of sodium a-naphthyl-phosphate in 0.0337 M Tris-borate buffer at pH 9.5. Preliminary studies suggested that there was no difference in the inhibition characteristics of alkaline phosphatase from tissue obtained at various times of
TABLE 1. Effect of Magnesium Ions and L-Phenylalanine on Human Endometrial, Intestinal, and Placental Alkaline Phosphatase Activity Phenol liberated" from phenyl phosphate, pH 10.1
Tissue Control
Mg2+, 5 mM
L-Phenylalanine, 5 rnM
J.LMlminlliter
Endometriunf Follicular phase Luteal phase Intestine Placenta
62.9 38.1 49.7 181.0
± ± ± ±
4.17 3.11 3.11 8.2
67.2 38.6 59.3 191.7
Results are the means of five observations ± standard deviations. bEndometrium was pooled from three subjects for each cycle phase.
a
± ± ± ±
6.29 1.72 3.63 7.81
44.6 25.9 16.7 81.7
± ± ± ±
4.25 2.58 2.84 4.3
\ Vol. 27, No.3
ENDOMETRIAL ALKALINE PHOSPHATASE
the cycle. Because of the small amount of enzyme activity present in late luteal phase tissue it was necessary to use pooled specimens. Endometrium was obtained, as above, from six more women and was divided into two pools. One pool was derived from three women who were between cycle days 10 and 16 and the other from three women between cycle days 22 and 25. The results reported for the inhibition studies are derived from the enzyme extracts from these pools, termed follicular phase and luteal phase tissue, respectively. The rate of hydrolysis of phenyl phosphate in the presence and absence of magnesium ions as magnesium chloride, at a concentration of 5 mM, was studied with a reaction mixture of 5 mM phenyl phosphate and 0.01 M 4-aminoantipyrine in a sodium carbonate-bicarbonate buffet' at pH 10.1. This formed the basic reaction mixture for the remaining studies. The effects of sodium deoxycholate, L-phenylalanine, and increasing concentrations of urea on the hydrolysis of phenyl phosphate by alkaline phosphatase derived from intestine, placenta, and endometrium were also studied. The rate of hydrolysis in the presence of 5 mM sodium deoxycholate was compared with that in control solutions containing no sodium deoxycholate. A similar reaction mixture was used to study the effects of 5 mM L-phenylalanine. Control solutions contained no phenylalanine. The concentration of urea required
301
to produce irreversible inactivation of phosphatase activity in the three types of tissue was determined. Tissue extracts were diluted with equal volumes of urea solutions to give final urea concentrations of 2,3,4,5, 6, 7, and 8 M. The solutions were allowed to stand at room temperature for 2 hours, at the end of which time the rate of phenyl phosphate hydrolysis was determined for each solution. RESULTS
Electrophoretic studies showed a single, constant band of enzyme activity in all 24 specimens. In particular, no difference was discernible between the single band of alkaline phosphatase in endometrium from late in the luteal phase, i.e., after cycle day 22, and that in the follicular phase tissue up to day 16. the endometrial enzyme appeared in a position anodal to the intestinal enzyme and cathodal to the placental enzyme. The results of the inhibition studies showed that endometrial alkaline phosphatase was slightly inhibited by L-phenylalanine, but not to the extent that the intestinal or placental enzymes were inhibited by this agent (Table 1). Magnesium ions had no significant effect on the endometrial or placental enzymes, although the activity of both enzymes was increased slightly in the presence of magnesium. The intestinal enzyme was increased to a greater extent in the presence of magnesium.
TABLE 2. Effect of Sodium Deoxycholate on Human Endometrial, Intestinal, and Placental Alkaline Phosphatase Activity Phenol liberated' from phenyl phosphate, pH 9.9 Tissue
Control
Sodium deoxycholate, 5 mM
% Control
/LMlminlliter
Endometrium" Follicular phase Luteal phase Intestine Placenta a Results
57.6 37.4 56.9 295.5
± 5.82 ± 2.81 ±
4.67
± 12.03
29.9 16.1 55.6 303.0
± 4.3
2.10 5.6 ± 20.27
are the means of five observations ± standard deviation. bEndometrium was pooled from three subjects for each cycle phase.
± ±
51.8 43 98 102.5
March 1976
WILSON
302
Sodium deoxycholate inhibited the endometrial enzyme but not the placental or intestinal enzymes (Table 2). Urea produced complete inhibition of the endometrial enzyme at a concentration of 3 M (Table 3). The intestinal enzyme was similarly inhibited by 5 M urea, while the placental enzyme was unaffected by 8 M urea. There were no differences in the enzyme derived from the luteal and follicular phases of the cycle with regard to Lphenylalanine or bile acid inhibition or inactivation by urea. DISCUSSION
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