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Histochemical demonstration of enzymes in the umbilical cord and membranes of human term pregnancy
EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1973,3/6,185-189.
EXCERPTA MEDICA
Histochemical demonstration of enzvmes in the umbilical cord and membraneiof human term pregnancy W. L. BENEDETTP, M. A. SALA AND H. ALVAREZ Laboratorio
de Investigaciones
sobre Reproduccidn
Humana,
Hospital
Pereira Rossell,
Montevideo,
Uruguay
BENEDETTI, W. L., SALA, M. and ALVAREZ, H. (1973): Histochemical demonstration of enzymes in the umbilicalcord and membranes of human term pregnancy. Europ. J. Obstet. Gynec. reprod. Biol., 3/6, 185-189. The enzymatic activities of fetal membranes and umbilical cord from normal term pregnancies were studied histochemically. One of the most outstanding results was the demonstration of some enzymes related to the steroid metabolism. This fact suggests that these ovular structures are able to participate in the synthesis of fetoplacental hormones. Predominance of the ‘slow-moving’ LDH isoenzymes over the ‘fast-moving’ ones, which could reflect a preponderance of anaerobic metabolism in both fetal annexes, is also demonstrated. umbilical cord; amnion; chorion; histochemistry
Introduction During the first stages of its development, the ovum is completely surrounded by the villous trophoblast. As its size increases, the villi related to the decidua capsularis become atrophic, and at four and a half months of amenorrhea, the trophoblast is constituted only by a few layers of cytotrophoblastic cells, among which are included the atrophic villi (chorion laeve). The villous trophoblast in contact with the decidua basalis grows and originates the placenta. The following layers can be observed in fetal membranes at term, from the amniotic cavity towards the decidua: _ the amniotic epithelium;
* Adjunto de Departamento Clinica Grynecotocologica Facultad de Medicina, Montevideo, Uruguay.
C,
- a layer of connective tissue, composed by there zones, two of which are derived from the amniotic and chorial mesoderm and, between these, a relatively acellular zone, derived from the extraembryonic coelom; - the trophoblast, constituted by three to eight rows of cytotrophoblastic cells, strongly attached to the maternal decidua. Despite their common origin with the placenta, the metabolic role of the fetal annexes during pregnancy is not well known. Lately, increasing attention is paid to amniotic fluid as a possible indicator of fetal condition. The possible participation of structures annexed to the fetus in the metabolism of the fetomaternal unit and in the mechanisms of fetomaternal interchange has also been stressed during the last years. Thus, the aim of this work is to study histochemically the enzymatic activities of the umbilical cord and fetal membranes at term, to further increase the knowledge of the functional role of these structures.
W. L. Benedetti
186
Material
and methods
Pieces of umbilical cord and fetal membranes (amnion and chorion) were obtained immediately after delivery from 15 normal term pregnancies (38-41 weeks of amenorrhea). The membranes were rolled to a tight cylinder, quickly frozen with Freon aerosol spray, and sectioned at 8 p with a cryostat at -20 “C. The sections were attached to slides by thawing, dried in a stream of air at room temperature during 3 minutes, and immediately incubated at 37 “C in the following media for the detection of enzymatic activities: - succinic dehydrogenase (SDH); - lactic dehydrogenase (LDH) and its fast-moving and slow-moving isoenzymes (Gerebtzoff, 1966); - NAD-linked secondary alcohol dehydrogenase, with isopropanol as substrate (Hardonk, 1965); - NAD-dependent 3/&hydroxysteroid dehydrogenase (3b-HDH), with dehydroepiandrosterone (DHA), androstenediol or pregnenediol as substrate; - NAD-linked 17&hydroxysteroid dehydrogenase (17,!?-HDH) with testosterone or 17/&estradiol as substrate; - monoamine oxydase (MAO); - aminopeptidase; - glucose-6-phosphatase (G6Pase) Controls for all these reactions were performed by incubation of sections in media without substrate. Detection of enzymatic activities in rat adrenal gland was used as control for steroid dehydrogenases. After incubation, the sections were fixed in 4% formalin for 24 h. Other sections of 8 p thickness were fixed for 18 h at 4 “C in 4% buffered formalin-sucrose at pH 7.2 and incubated at 37 “C, after thorough rinsing, in the following media for the demonstration of enzymatic activities: - alkaline phosphatase, with the Gomori-Takamatsu method and the Burstone simultaneous coupling azo dye technique; - acid phosphatase, after Gomori; - 5-nucleotidase, according to Wachstein and Meisel (1957); _ adenosine triphosphatase (ATPase) after Wachstein and Meisel; - thiamine pyrophosphatase (TPPase) according to Novikoff and Goldfischer (1961);
et al., Histochemistry
of umbilical
cord and membranes
at term
- nonspecific esterase, with the Burstone naph-
thol AS-acetate method. Lipids were stained with Sudan Black B or with Oil Red 0. The histochemical methods were performed following instructions of Barka and Anderson (1965), unless otherwise indicated. After washing, all sections were mounted in glycerin jelly. Sections routinely stained with hematoxylin and eosin, and with periodic acid-Schiff(PAS)-hematoxylin were used as morphological controls. Results
Table I shows the semiquantitative evaluation of different enzymatic activities in the umbilical cord and membranes. The single epithelial sheet lining the umbilical cord has a very intense activity of LDH and its slow-moving isoenzymes, and a strong 17P-HDH activity, with both substrates employed. The connective cells of the umbilical cord exhibit strong positive reactions for all dehydrogenases, except the succinic one, which shows weak activity. Enzymatic activities in the umbilical vessels are very intense for total LDH and its slow-moving isoenzymes, TPPase and ATPase (Fig. l), lower for LDH fast-moving isoenzymes and SDH, and weak for steroid dehydrogenases and nonspecific esterases. Only traces of alkaline and acid-phosphatase activity are demonstrated in the endothelium of umbilical vessels and for 5-nucleotidase in the muscular layer.
Fig. 1.
Umbilical muscular
vein. Intense layer (x 60).
activity
in ATPase
in the
W. L. Benedetti et al., Histochemistry
TABLE I
of umbilical cord and membranes ht term
187
Distribution of enzymaticactivitiesin human umbilicalcord and fetal membranes --
~_
-
Umbilical
cord
Fetal membranes
Enzymes
Alkaline phosphatases
_
( ~~r~o~,
Acid phosphatases 5-nucleotidase ATPase TPPase G6Pase MAO SDH LDH LDH (slow-moving isoenzymes) LDH (fast-moving isoenzymes) Alcohol DH DHA androstenediol 3/3-HDH pregnenediol testosterone “psHDH 1 178-estradiol Amino peptidase Esterase Lipids - No activity; I_t doubtful;
+ weak; t+
I -
It i fii +++ i + ++ +-t+ +++ ++ i + + It + + i + i
_
I -t ++
Sf ‘f
t I + + +
intense; $++
There is intense activity in the amniotic epithelium for total LDH and its slow-moving isoenzymes, lower for the fast-moving ones and SDH, weak for the other enzymes studied. Several lipid droplets of various sizes are found in the amniotic epithelium, as well as in the umbilical cord epithelium. The connective tissue cells of the chorio-amniotic membrane present weak activities for MAO, SDH, and fast-moving LDH isoenzymes, while the activities for the other dehydrogenases and aminopeptidase are strong. No hydrolytic enzymes are demonstrated. The trophoblastic layer of the chorion shows very intense activity for total LDH and its slow-moving isoenzymes (Figs. 2a and 2b), and for ATPase. The 3/?-HDH activity is higher when DHA was used as substrate. The 17/?-HDH activity is similarly intense with both substrates used for its demon-
‘+ ++ ++ ++ ++ f-t ++ ++ + * + +-
ItI It It + + + *
++ +t + +i +++ ii
4
5+
++
++ t i- +
+i+ +- +
+
+++
++
++
+ i
+ i+++
+
++
i
++
+
++
+
++
+
++
IA++
+ + -t- $
very intense
stration (Fig. 3). The remaining enzymes studied show strong activities, except the G6Pase, acid phosphatase and esterase. Several lipid droplets are also found in the trophoblastic layer. No enzymatic activities are found in atrophic villi, in contrast to the strong activities present in the trophoblastic layer of the chorion laeve that surrounds them.
Discussion
Scarce bibliography on the histochemistry of the fetal annexes is available. This is usually related to specific structures (e.g. amniotic epithelium) or to one particular enzymatic group. Our observations on the enzymatic activities of the umbilical vessels agree in general with previous
188
W. L. Benedetti et al., Histochemistry of umbilical cord and membranes at term
Fig. 3. Fetal membranes. 17B-HDH activity demonstrated with testosterone as substrat, and NAD as cofactor (X 100).
LDH-activity demonstrated in fetal membranes Fig. 2. (x 50).a.slow-moving isoenzymes; b. fast-moving isoenzymes.
studies (Krauer and Ludwig, 1968) although we have demonstrated an activity in 3/3-HDH, which has not been observed before. The several lipids found in fetal membranes raise a question as to their origin. The absorption of lipids from the amniotic fluid seems unlikely due to the absence of hydrolytic enzymes. Pritchard, Armstrong and Wilt (1968) demonstrated that fatty acids can be esterified by membrane slices into triglycerides and cholesterol esters. In vivo, precursors for the biosynthesis are probably obtained from maternal circulation and/or by synthesis in situ. These lipids could be used in the metabolism of membranes and, in view of their similar composition, could also be released into the amniotic cavity (Pritchard et al., 1968).
Biochemically it has been demonstrated that LDH activity is higher in membranes than in the placenta (Lapan and Friedman, 1965). We have demonstrated an intense activity of LDH in these structures by histochemistry; such activity is mainly due to the slow-moving isoenzymes, as in the villous trophoblast of the human placenta (Benedetti, Sala, Kulay and Alvarez, 1971). The more intense enzymatic activities are found in the chorionic trophoblast. The 3B-HDH activity is higher when DHA is used as substrate, as in the villous trophoblast (Benedetti and Alvarez, 1967). This agrees with the finding that in the fetoplacental unit the steroid biosynthesis is mainly carried out through DHA (Diczfalusy, 1968). The 17/3-HDH activity is demonstrated both with testosterone and estradiol as substrate, and NAD as cofactor. Mitsudo and Koide (1966) had already demonstrated its activity only with estradiol as a substrate and NAD as a cofactor, but neither with testosterone as a substrate, nor using NADP as a cofactor. During the first half of pregnancy, the activity of 17P-HDH in the chorial trophoblast is comparatively higher than in adrenals and testis of the human fetus of the same gestational age (Sulcova, Capkova and Starka, 1970). High 17B-HDH activity in cells of the human amnion and chorion was demonstrated with perfusion techniques by Gurpide, Stolee and Tseng (1971).
W. L. Benedetti
et al., Hisiochemistry
of umbilical
cord and membranes
The following observations suggest that the membranes and umbilical cord participate together with the placental trophoblast in steroid biosynthesis: intense aminopeptidase activity, related to such metabolism (Jones, Goldberg and Woodruff, 1968); presence of secondary alcohol dehydrogenase, that would catalyze the cleavage of the cholesterol side chain for its conversion into pregnenolone (Hardonk, 1965); and the high steroid dehydrogenase activities. According to the results available up to now, the ovular membranes and umbilical cord must not be considered merely as simple structures assigned to contain and mechanically support the fetus or to carry blood between fetus and placenta. The histological and ultrastructural complexity of these structures and their abundant and intense enzymatic activities evidence their functional role in fetal homeostasis and in hormonal, mainly steroidal synthesis of human pregnancy. References Barka, T. and Anderson, P. J. (1965): Histochemistry. Harper and Row, New York, N. Y. Benedetti, W. L. and Alvarez, H. (1967): Histoquimica de la placenta humana en distintas edades de gravidez. Obstet. Ginec. lat.-amer., 25, 387. Benedetti, W. L., Sala, M. A., Kulay Jr, L. and Alvarez, H. (1971): Histochemical localization of lactic dehydrogenase isoenzymes in the human placenta. Amer. J. Obstet. Gynec., 110, 742. Diczfalusy, E. (1968): Steroid metabolism in the foetoplacental unit. In: Proceedings, International Symposium on the Foeto-placental Unit, Milan 1968. ICS 183, Excerpta Medica, Amsterdam.
at term
Gerebtzoff, enzymes ganglion
189 M. A. (1966): Detection histochimique d’isode la lactate deshydrogenase dans le nerf et le spinal. C. R. Sot. Biol. (Paris), 160, 1323.
Gurpide, E., Stolee, A. and Tseng, L. (1971): Quantitative studies of tissue uptake and disposition of hormones. Acta endocr. (Kbh.), 67, Suppl. 153, 247. Hardonk, M. J. (1965): A new method for the histochemical demonstration of steroid producing cell in human tissues. Histochemie, 5, 234. Jones, G. E. S., Goldberg, B. and Woodruff, Histochemistry as a guide for interpretation tion. Amer. J. Obstet. Gynec., 100, 76.
J. D. (1968): of cell func-
Krauer, F. and Ludwig, K. S. (1968): Enzymhistochemische Beobachtungen an den fetalen Gefassen geburtsreifer menschlicher Plazenten. Gynaecologia (Base& 166, 162. Lapan, B. and Friedman, M. M. (1965): Tissue enzymes in gestation: comparative activities in the placenta and fetal membranes. Amer. J. Obstet. Gynec., 93, 1157. Mitsudo, S. M. and Koide, S. S. (1966): Histochemical demonstration of the hydroxysteroid oxidoreductases in the umbilical cord and membranes of human term and immature placenta. Endocrinology, 78, 333. Novikoff, A. B. and Goldfischer, S. (1961): Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. froc. nnt. Acad. Sci. (Wash.), 47, 802. Pritchard, E. T., Armstrong, W. D. and Wilt, J. C. (1968): Examination of lipids from amnion, chorion and vernix. Amer. J. Obstet. Gynec., 100, 289. Sulcova, J., Capkova, A. and Starka, L. (1970): 178hydroxysteroid dehydrogenase activity in human foetal adrenals, gonads and chorion from the first half of foetal life. Endow. exp.. 4, 83. Wachstein, M. and Meisel, E. (1957): hepatic phosphatases at a physiologic Pathol., 27, 13.
Histochemistry of pH. Amer. J. clin.
EXCERPTA MEDICA
Histochemical demonstration of enzvmes in the umbilical cord and membraneiof human term pregnancy W. L. BENEDETTP, M. A. SALA AND H. ALVAREZ Laboratorio
de Investigaciones
sobre Reproduccidn
Humana,
Hospital
Pereira Rossell,
Montevideo,
Uruguay
BENEDETTI, W. L., SALA, M. and ALVAREZ, H. (1973): Histochemical demonstration of enzymes in the umbilicalcord and membranes of human term pregnancy. Europ. J. Obstet. Gynec. reprod. Biol., 3/6, 185-189. The enzymatic activities of fetal membranes and umbilical cord from normal term pregnancies were studied histochemically. One of the most outstanding results was the demonstration of some enzymes related to the steroid metabolism. This fact suggests that these ovular structures are able to participate in the synthesis of fetoplacental hormones. Predominance of the ‘slow-moving’ LDH isoenzymes over the ‘fast-moving’ ones, which could reflect a preponderance of anaerobic metabolism in both fetal annexes, is also demonstrated. umbilical cord; amnion; chorion; histochemistry
Introduction During the first stages of its development, the ovum is completely surrounded by the villous trophoblast. As its size increases, the villi related to the decidua capsularis become atrophic, and at four and a half months of amenorrhea, the trophoblast is constituted only by a few layers of cytotrophoblastic cells, among which are included the atrophic villi (chorion laeve). The villous trophoblast in contact with the decidua basalis grows and originates the placenta. The following layers can be observed in fetal membranes at term, from the amniotic cavity towards the decidua: _ the amniotic epithelium;
* Adjunto de Departamento Clinica Grynecotocologica Facultad de Medicina, Montevideo, Uruguay.
C,
- a layer of connective tissue, composed by there zones, two of which are derived from the amniotic and chorial mesoderm and, between these, a relatively acellular zone, derived from the extraembryonic coelom; - the trophoblast, constituted by three to eight rows of cytotrophoblastic cells, strongly attached to the maternal decidua. Despite their common origin with the placenta, the metabolic role of the fetal annexes during pregnancy is not well known. Lately, increasing attention is paid to amniotic fluid as a possible indicator of fetal condition. The possible participation of structures annexed to the fetus in the metabolism of the fetomaternal unit and in the mechanisms of fetomaternal interchange has also been stressed during the last years. Thus, the aim of this work is to study histochemically the enzymatic activities of the umbilical cord and fetal membranes at term, to further increase the knowledge of the functional role of these structures.
W. L. Benedetti
186
Material
and methods
Pieces of umbilical cord and fetal membranes (amnion and chorion) were obtained immediately after delivery from 15 normal term pregnancies (38-41 weeks of amenorrhea). The membranes were rolled to a tight cylinder, quickly frozen with Freon aerosol spray, and sectioned at 8 p with a cryostat at -20 “C. The sections were attached to slides by thawing, dried in a stream of air at room temperature during 3 minutes, and immediately incubated at 37 “C in the following media for the detection of enzymatic activities: - succinic dehydrogenase (SDH); - lactic dehydrogenase (LDH) and its fast-moving and slow-moving isoenzymes (Gerebtzoff, 1966); - NAD-linked secondary alcohol dehydrogenase, with isopropanol as substrate (Hardonk, 1965); - NAD-dependent 3/&hydroxysteroid dehydrogenase (3b-HDH), with dehydroepiandrosterone (DHA), androstenediol or pregnenediol as substrate; - NAD-linked 17&hydroxysteroid dehydrogenase (17,!?-HDH) with testosterone or 17/&estradiol as substrate; - monoamine oxydase (MAO); - aminopeptidase; - glucose-6-phosphatase (G6Pase) Controls for all these reactions were performed by incubation of sections in media without substrate. Detection of enzymatic activities in rat adrenal gland was used as control for steroid dehydrogenases. After incubation, the sections were fixed in 4% formalin for 24 h. Other sections of 8 p thickness were fixed for 18 h at 4 “C in 4% buffered formalin-sucrose at pH 7.2 and incubated at 37 “C, after thorough rinsing, in the following media for the demonstration of enzymatic activities: - alkaline phosphatase, with the Gomori-Takamatsu method and the Burstone simultaneous coupling azo dye technique; - acid phosphatase, after Gomori; - 5-nucleotidase, according to Wachstein and Meisel (1957); _ adenosine triphosphatase (ATPase) after Wachstein and Meisel; - thiamine pyrophosphatase (TPPase) according to Novikoff and Goldfischer (1961);
et al., Histochemistry
of umbilical
cord and membranes
at term
- nonspecific esterase, with the Burstone naph-
thol AS-acetate method. Lipids were stained with Sudan Black B or with Oil Red 0. The histochemical methods were performed following instructions of Barka and Anderson (1965), unless otherwise indicated. After washing, all sections were mounted in glycerin jelly. Sections routinely stained with hematoxylin and eosin, and with periodic acid-Schiff(PAS)-hematoxylin were used as morphological controls. Results
Table I shows the semiquantitative evaluation of different enzymatic activities in the umbilical cord and membranes. The single epithelial sheet lining the umbilical cord has a very intense activity of LDH and its slow-moving isoenzymes, and a strong 17P-HDH activity, with both substrates employed. The connective cells of the umbilical cord exhibit strong positive reactions for all dehydrogenases, except the succinic one, which shows weak activity. Enzymatic activities in the umbilical vessels are very intense for total LDH and its slow-moving isoenzymes, TPPase and ATPase (Fig. l), lower for LDH fast-moving isoenzymes and SDH, and weak for steroid dehydrogenases and nonspecific esterases. Only traces of alkaline and acid-phosphatase activity are demonstrated in the endothelium of umbilical vessels and for 5-nucleotidase in the muscular layer.
Fig. 1.
Umbilical muscular
vein. Intense layer (x 60).
activity
in ATPase
in the
W. L. Benedetti et al., Histochemistry
TABLE I
of umbilical cord and membranes ht term
187
Distribution of enzymaticactivitiesin human umbilicalcord and fetal membranes --
~_
-
Umbilical
cord
Fetal membranes
Enzymes
Alkaline phosphatases
_
( ~~r~o~,
Acid phosphatases 5-nucleotidase ATPase TPPase G6Pase MAO SDH LDH LDH (slow-moving isoenzymes) LDH (fast-moving isoenzymes) Alcohol DH DHA androstenediol 3/3-HDH pregnenediol testosterone “psHDH 1 178-estradiol Amino peptidase Esterase Lipids - No activity; I_t doubtful;
+ weak; t+
I -
It i fii +++ i + ++ +-t+ +++ ++ i + + It + + i + i
_
I -t ++
Sf ‘f
t I + + +
intense; $++
There is intense activity in the amniotic epithelium for total LDH and its slow-moving isoenzymes, lower for the fast-moving ones and SDH, weak for the other enzymes studied. Several lipid droplets of various sizes are found in the amniotic epithelium, as well as in the umbilical cord epithelium. The connective tissue cells of the chorio-amniotic membrane present weak activities for MAO, SDH, and fast-moving LDH isoenzymes, while the activities for the other dehydrogenases and aminopeptidase are strong. No hydrolytic enzymes are demonstrated. The trophoblastic layer of the chorion shows very intense activity for total LDH and its slow-moving isoenzymes (Figs. 2a and 2b), and for ATPase. The 3/?-HDH activity is higher when DHA was used as substrate. The 17/?-HDH activity is similarly intense with both substrates used for its demon-
‘+ ++ ++ ++ ++ f-t ++ ++ + * + +-
ItI It It + + + *
++ +t + +i +++ ii
4
5+
++
++ t i- +
+i+ +- +
+
+++
++
++
+ i
+ i+++
+
++
i
++
+
++
+
++
+
++
IA++
+ + -t- $
very intense
stration (Fig. 3). The remaining enzymes studied show strong activities, except the G6Pase, acid phosphatase and esterase. Several lipid droplets are also found in the trophoblastic layer. No enzymatic activities are found in atrophic villi, in contrast to the strong activities present in the trophoblastic layer of the chorion laeve that surrounds them.
Discussion
Scarce bibliography on the histochemistry of the fetal annexes is available. This is usually related to specific structures (e.g. amniotic epithelium) or to one particular enzymatic group. Our observations on the enzymatic activities of the umbilical vessels agree in general with previous
188
W. L. Benedetti et al., Histochemistry of umbilical cord and membranes at term
Fig. 3. Fetal membranes. 17B-HDH activity demonstrated with testosterone as substrat, and NAD as cofactor (X 100).
LDH-activity demonstrated in fetal membranes Fig. 2. (x 50).a.slow-moving isoenzymes; b. fast-moving isoenzymes.
studies (Krauer and Ludwig, 1968) although we have demonstrated an activity in 3/3-HDH, which has not been observed before. The several lipids found in fetal membranes raise a question as to their origin. The absorption of lipids from the amniotic fluid seems unlikely due to the absence of hydrolytic enzymes. Pritchard, Armstrong and Wilt (1968) demonstrated that fatty acids can be esterified by membrane slices into triglycerides and cholesterol esters. In vivo, precursors for the biosynthesis are probably obtained from maternal circulation and/or by synthesis in situ. These lipids could be used in the metabolism of membranes and, in view of their similar composition, could also be released into the amniotic cavity (Pritchard et al., 1968).
Biochemically it has been demonstrated that LDH activity is higher in membranes than in the placenta (Lapan and Friedman, 1965). We have demonstrated an intense activity of LDH in these structures by histochemistry; such activity is mainly due to the slow-moving isoenzymes, as in the villous trophoblast of the human placenta (Benedetti, Sala, Kulay and Alvarez, 1971). The more intense enzymatic activities are found in the chorionic trophoblast. The 3B-HDH activity is higher when DHA is used as substrate, as in the villous trophoblast (Benedetti and Alvarez, 1967). This agrees with the finding that in the fetoplacental unit the steroid biosynthesis is mainly carried out through DHA (Diczfalusy, 1968). The 17/3-HDH activity is demonstrated both with testosterone and estradiol as substrate, and NAD as cofactor. Mitsudo and Koide (1966) had already demonstrated its activity only with estradiol as a substrate and NAD as a cofactor, but neither with testosterone as a substrate, nor using NADP as a cofactor. During the first half of pregnancy, the activity of 17P-HDH in the chorial trophoblast is comparatively higher than in adrenals and testis of the human fetus of the same gestational age (Sulcova, Capkova and Starka, 1970). High 17B-HDH activity in cells of the human amnion and chorion was demonstrated with perfusion techniques by Gurpide, Stolee and Tseng (1971).
W. L. Benedetti
et al., Hisiochemistry
of umbilical
cord and membranes
The following observations suggest that the membranes and umbilical cord participate together with the placental trophoblast in steroid biosynthesis: intense aminopeptidase activity, related to such metabolism (Jones, Goldberg and Woodruff, 1968); presence of secondary alcohol dehydrogenase, that would catalyze the cleavage of the cholesterol side chain for its conversion into pregnenolone (Hardonk, 1965); and the high steroid dehydrogenase activities. According to the results available up to now, the ovular membranes and umbilical cord must not be considered merely as simple structures assigned to contain and mechanically support the fetus or to carry blood between fetus and placenta. The histological and ultrastructural complexity of these structures and their abundant and intense enzymatic activities evidence their functional role in fetal homeostasis and in hormonal, mainly steroidal synthesis of human pregnancy. References Barka, T. and Anderson, P. J. (1965): Histochemistry. Harper and Row, New York, N. Y. Benedetti, W. L. and Alvarez, H. (1967): Histoquimica de la placenta humana en distintas edades de gravidez. Obstet. Ginec. lat.-amer., 25, 387. Benedetti, W. L., Sala, M. A., Kulay Jr, L. and Alvarez, H. (1971): Histochemical localization of lactic dehydrogenase isoenzymes in the human placenta. Amer. J. Obstet. Gynec., 110, 742. Diczfalusy, E. (1968): Steroid metabolism in the foetoplacental unit. In: Proceedings, International Symposium on the Foeto-placental Unit, Milan 1968. ICS 183, Excerpta Medica, Amsterdam.
at term
Gerebtzoff, enzymes ganglion
189 M. A. (1966): Detection histochimique d’isode la lactate deshydrogenase dans le nerf et le spinal. C. R. Sot. Biol. (Paris), 160, 1323.
Gurpide, E., Stolee, A. and Tseng, L. (1971): Quantitative studies of tissue uptake and disposition of hormones. Acta endocr. (Kbh.), 67, Suppl. 153, 247. Hardonk, M. J. (1965): A new method for the histochemical demonstration of steroid producing cell in human tissues. Histochemie, 5, 234. Jones, G. E. S., Goldberg, B. and Woodruff, Histochemistry as a guide for interpretation tion. Amer. J. Obstet. Gynec., 100, 76.
J. D. (1968): of cell func-
Krauer, F. and Ludwig, K. S. (1968): Enzymhistochemische Beobachtungen an den fetalen Gefassen geburtsreifer menschlicher Plazenten. Gynaecologia (Base& 166, 162. Lapan, B. and Friedman, M. M. (1965): Tissue enzymes in gestation: comparative activities in the placenta and fetal membranes. Amer. J. Obstet. Gynec., 93, 1157. Mitsudo, S. M. and Koide, S. S. (1966): Histochemical demonstration of the hydroxysteroid oxidoreductases in the umbilical cord and membranes of human term and immature placenta. Endocrinology, 78, 333. Novikoff, A. B. and Goldfischer, S. (1961): Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. froc. nnt. Acad. Sci. (Wash.), 47, 802. Pritchard, E. T., Armstrong, W. D. and Wilt, J. C. (1968): Examination of lipids from amnion, chorion and vernix. Amer. J. Obstet. Gynec., 100, 289. Sulcova, J., Capkova, A. and Starka, L. (1970): 178hydroxysteroid dehydrogenase activity in human foetal adrenals, gonads and chorion from the first half of foetal life. Endow. exp.. 4, 83. Wachstein, M. and Meisel, E. (1957): hepatic phosphatases at a physiologic Pathol., 27, 13.
Histochemistry of pH. Amer. J. clin.