The Journal of Obstetrics and Gynaecology of the British Commonwealth NOV. 1971. V O ~ . 78. pp. 981-986.

A QUANTITATIVE STUDY OF THE GLYCOGEN CONTENT OF HUMAN FETAL SKIN IN THE FIRST TRIMESTER

BY FRANK SHARP, Lecturer

Department o j Obstetrics, University of‘ Glasgow

Summary Using a quantitative colorimetric micromethod for the determination of glycogen in tissues, the pattern of glycogen reserves in whole thickness human fetal skin between the 8th and the 16th weeks of gestation has been established, and com- pared with that of other fetal tissues in the same gestational range. The value of the results is discussed, and speculation made as to the role of glycogen in human fetal skin, and its relationship to the general carbohydrate economy of the fetus.

MORE than one hundred years ago Claude Bernard demonstrated glycogen in fetal tissues and described its distribution at varying periods of gestation. Following this work it was con- sidered that many fetal tissues were rich in glycogen, and that it played some role in growth and differentiation processes. After this early work, which was chiefly histochemical (Cremer, 1902; Needham, 193 I ) , quantitative analytical methods developed and more accurate studies of the glycogen content of various fetal tissues were made. (See review by Shelley, 1961.)

Bernard (1859, 1878), who first devised a histochemical technique for the demonstration of glycogen in tissues, included brief reference to the glycogen in skin. He found the substance in the fetal epidermis of the pig, lamb, cat and calf, and-related its absence in the adult organism to the “organization” of the tissue and keratiniza- tion. In the early human fetus all the cutaneous appendages, including the epidermis, sebaceous and sweat glands, and hair follicles, are rich in glycogen (Montagna, 1965). Serri and Huber ( 1963) summarized the development of human fetal epidermis and cutaneous appendages, and described the location of glycogen in these structures, at a cellular level, as demonstrated histochemically. Early in the first trimester (from the third week of fetal life) the epidermis is represented by a single layer of undifferentiated

cells, filled with glycogen. By the fourth week the epidermis has become two layered, with an outer periderm or epitrichial layer and a basal or germinative layer, containing glycogen through- out. During the tenth to the twelfth weeks, the basal layer proliferates to add an intermediate layer, the cells of which also contain glycogen. The intermediate layer becomes stratified to form a spinous layer by approximately the 13th to 16th week. These cells also are rich in glycogen. but by this stage the cells of the basal layer are almost entirely free of P.A.S. positive substance. Coincidently with these changes, the develop- ment of the hair follicles proceeds. At nine weeks the hair germs form from the basal layer. The cells of the hair germ are glycogen-free. By 16 weeks or so each hair germ has become a pilosebaceous unit. Within this unit the cells of the arrector pilorum “bulge” are rich in glycogen. The anlage of the sebaceous gland is composed of cells containing a moderate amount of glycogen, but with cellular differentiation and functional development, as lipid droplets form in the central cells these lose their glycogen, and this becomes limited to the peripheral acinar cells. Furthermore, by this stage large amounts of glycogen are demonstrable in the ground substance of the dermis, the presumptive hypodermis and the fatty layer.

The large body of histochemical work on the 981

982 SHARP

glycogen content of fetal tissues generally is in good agreement with the results of quantitative chemical analyses. For the human fetus quantita- tive data is available for glycogen in the following tissues : liver, lung, heart, skeletal muscle, brain, kidney and placenta (Shelley, 1961). However, no such quantitative study has yet been made using human fetal skin, and this was the object of the present study covering the first 16 weeks of gestation.

MATERIALS The fetal material was obtained from fresh

abortuses collected at the time of therapeutic termination of pregnancy, which was undertaken for various indications, There was no reason to suspect fetal abnormality in any case. In the majority of cases termination was performed surgically by the vaginal route. In the remaining small number the fetus with the complete gestation sac was collected at the time of hysterotomy. With all the material, therefore, complete freshness was guaranteed, and in no case had there been any likely antecedent period of fetal hypoxia.

The following tissues were sought: ( I ) Skin. This was dissected in whole thick-

ness from either the trunk or the limbs with the aid of a fixed magnifier (approximately x5) and fine instruments. With the smaller fetuses an initial pilot study was performed by comparing the glycogen content of the dis- sected “whole thickness skin” and that of the immediately underlying muscle tissue. A significant difference was noted, and it was concluded that “whole thickness skin” ob- tained in this fashion did not contain elements of muscle. With larger fetuses the plane of separation between skin and underlying tissues was easy to define.

(2) In a smaller number of cases the follow- ing were collected as control tissues, in which the pattern of glycogen reserves was already known: liver, heart, lung, kidney, adrenal gland, and skeletal muscle.

METHODS For the quantitative estimation the method of

Kemp and Kits van Heijningen (1954) was used. This is a colorimetric micro-method for the

determination of glycogen in tissues as total carbohydrate, and depends on a colour reaction which occurs when a dilute solution of glucose is heated with concentrated sulphuric acid. Glycogen is hydrolyzed to glucose in hot sulphuric acid, and therefore the reaction can be used to determine glycogen. Any glucose present in the tissue will also be determined, but the amount of free glucose present in tissues is usually small compared to the glycogen present, and its contribution was negligible in the present study.

Reagents Deproteinizing solution. Trichloroacetic acid

( 5 g., A.R.) and silver sulphate (100 mg., A.R.), dissolved in water and made up to 100 ml. The solution was stored in an amber bottle in the cold.

Sulphuric acid, 96 per cent (w/w, approxi- mately 36 N.).

Procedure The freshly collected tissue was freed from any

adherent blood clot and gently washed with physiological saline solution to remove obvious contamination with fluid blood. The tissue (20-70 mg.) was then weighed accurately, and ground with 5 ml. of the deproteinizing solution, boiled for 15 minutes, and cooled in running water. After making up to 5 ml. again with deproteinizing solution the preparation was centrifuged at 3000 revolutions per minute for 5 minutes. One ml. of the clear supernatant solution was then added to 3 ml. of sulphuric acid and mixed by vigorom shaking. This mixture was then heated in a boiling water bath for exactly 6 .5 minutes, and then cooled in running water. The intensity of the pink colour produced was measured spectrophotometrically (Spectromom, Budapest), at 490, 520 and 550 mw. A corrected extinction was then calculated by using E520 - Elno+E,,,, where Ex was the

( 2 ) extinction at the respective wavelengths (Allen, 1950). The glycogen concentration was then read from a standard curve prepared from solutions containing known concentrations of glucose. Finally, the glycogen content of the given tissue was calculated as total carbohydrate,

GLYCOGEN CONTENT OF HUMAN FETAL SKIN IN THE FIRST TRIMESTER 983

in mg. glucose per g. wet weight. The intensity of the pink colour is proportional to the amount of glucose up to a concentration of 150 pg. per ml. In the case of cardiac tissue, where the concentration of glucose in the deproteinized extract was usually greater than 150 pg. per ml., a suitably diluted solution was prepared before the colour reaction was carried out.

In a small number of cases representative of various stages in the first trimester, tissue specimens were collected for parallel histo- chemical studies, using the periodic acid-Schiff reaction.

RESULTS Two hundred and nine specimens were assayed

quantitatively for glycogen, covering the 8th to the 16th weeks of gestation as calculated from the last menstrual period. Of these, 92 were skin specimens, and other tissues examined are detailed in Table I . Figures 1-6 show the pattern of the glycogen reserves in the seven tissues under consideration (skin, liver, heart, skeletal muscle, lung, kidney, and adrenal gland), relating the values as total carbohydrate (mg. glucose per g. wet weight) to gestational age. Table 11 sum- marizes the calculated mean values for each tissue at two-week intervals.

TABLE I Glycogen reserves in fetal tissues Numbers of specimens examined

Tissue

Skin .. _ . Liver . , . . Heart . . . . Lung . . .. Skeletal muscle Kidney . . Adrenal . .

Weeks gestation

12 14 16

26 15 10 6 6 5 9 6 7 7 6 7 5 6 5 5 4 5 2 2 -

The principal interest in this study was the skin glycogen, and the 92 specimens represent nearly half of the total number of assays per- formed. The mean values in skin fluctuated little, and varied from 7-7-10.5 mg. glucose per g. wet weight of whole thickness skin. (Table I1 and Fig. I ) . The mean values in liver (Table 11) were

HUMAN FETAL SKIN

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0 0. 0

6 6 8 10 12 14 16

WEEKS GESTATION

FIG. 1 Glycogen reserves in human fetal skin measured as mg.

glucose per g. wet weight.

TABLE I1 Glycogen reserves in fetal tissues

Mean values measured as total carbohydrate (mg. glucose/gram wet weight tissue)

~

Weeks gestation

8 10 12 14 16 Tissue

Skin .. .. 10.2 10.5 10.5 10.4 7 .7 Liver . . . . 3 .7 3 .5 3 .8 8 . 5 15.1 Heart . . . . - 68-8 45 .0 60 .2 63.5 Lung _ . .. - 13.9 13-7 13.6 16.3 Skeletal muscle - 15.1 17.0 18.4 19.2 Kidney . . - - 3 . 4 3 . 3 4 . 2 Adrenal .. - - 3-1 2 - 3 -

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lower in the major portion of the first trimester (3.5-3.8 mg. glucose per g. wet weight) but rose to 15.1 mg. glucose per g. wet weight by the 16th week (Fig. 2). Values for cardiac tissue (Fig. 3) were the highest in the tissues examined, with means ranging from 45.0 to 6 8 . 8 mg. glucose per g. wet weight. In skeletal muscle (Fig. 4) the mean values showed a gradual increase from 15.1 at ten weeks to 19.2 mg. glucose per g. wet weight at sixteen weeks. Initially of the order 13-6-13-9 mg. glucose per g. wet weight, the mean value in lung rose to 16.3 mg. glucose per g. wet weight at 16 weeks (Fig. 5). Whole kidney showed little variance in the samples assayed, with mean values 3.3- 4.1 mg. glucose per g. wet weight at 12-1 6 weeks

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WEEKS GESTATION FIG. 2

Glycogen reserves in human fetal liver measured as mg. glucose per g. wet weight.

HUMAN FETAL HEART

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WEEKS GESTATION

FIG. 3 Glycogen reserves in human fetal heart measured as rng.

glucose per g. wet weight.

(Fig. 6). Whole adrenal gland contained the least glycogen of all the tissues examined, with mean values at 2.3-3.1 mg. glucose per g. wet weight (Fig. 6) .

The parallel histochemical studies, using the periodic acid-Schiff method for the localization of the glycogen at a cellular level, confirmed the findings of earlier workers, particularly the pattern in skin which has been described fully in the introduction.

DISCUSSION The principal object of this study was to

establish quantitatively the pattern of glycogen reserves in human fetal skin in the first trimester. No similar quantitative study has previously

GLYCOGEN CONTENT OF HUMAN FETAL SKIN IN THE FIRST TRIMESTER 985

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been described. As all the tissues were completely fresh and assayed immediately glycogen values likely to represent the true levels before the moment of death of the fetus. No known ante- cedent period of significant fetal hypoxia had occurred in any case which might have caused mobilization of tissue glycogen. These last two factors may explain the higher mean values obtained in the control tissues in this study compared with the values previously reported (Szendi, 1936; Villee, 1954). Furthermore, in this study the assay of total tissue carbohydrate was used as a measure of tissue glycogen. Good agreement was obtained between the values for total carbohydrate and glycogen in the liver and skeletal muscle of fetal dogs (Schlossman, 1938), and Shelley (1961) considered that the measure-

HUMAN FETAL LUNG

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Glycogen reserves in human fetal skeletal muscle measured as mg. glucose per g. wet weight.

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986 SHARP

to contain noteworthy quantities in the first trimester. How these local glycogen reserves in the skin relate to the carbohydrate economy of the fetus generally is still a matter for specula- tion. That the skin glycogen may be an available store of carbohydrate for the fetus is one possibility. On the other hand the high local

0 . M - MEAN VALUES(KIDNEY) -o----o- MEAN VALUES (ADRENAL)

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3 3 (3 8 10 12 14 16

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Glycogen reserves in human fetal kidney and adrenal gland, measured as mg. glucose per g. wet weight.

concentration in fetal skin in the first trimester may be related to chemical transfer activity between the fetus and the liquor amnii.

ACKNOWLEDGEMENTS I thank Professor J. A. Milne, Department of

Dermatology, University of Glasgow, for en- couragement and invaluable advice throughout this work. This study was carried out while a guest research worker in the department of Professor F. E. Szontagh, Professor of Obstetrics and Gynaecology, the Medical University of Szeged, Hungary, with the aid of a Travelling Scholarship from the William McCunn Trust. I am indebted also to Dr. G . Varga, Department of Pathology, Szeged, who assisted with the histochemical preparations.

REFERENCES Allen, W. M. (1950): Journal of Clinical Endocrinology

and Metabolism, 10, 71. Bernard, C. (1859): Comptes rendus hebdomadaires des

skances de I’Acadkmie des sciences, 48, 673. Bernard, C. (1878): Lecons sur les phenomenes de la vie

communs aux animaux et aux vegetacrx. Vol. 2. J. B. Bailliere et Fils, Paris. p. 72.

Cremer, M. (1902) : Ergebnisse der Physiologie, biologi- schen Chemie und experimentellen Pharmakologie, 1, 803.

Kemp, A,, and Kits van Heijningen, A. J. M. (1954): Biochemical Journal, 56, 646.

Mendel, B., Kemp, A., and Meyers, D. K. (1954): Biochemical Journal, 56, 639.

Montagna, W. (1956): The Structure and Function of Skin, 1st edition. Academic Press, New York, p. 52.

Needham, J. (1931): Chemical Embryology, vol. 2, section 8. University Press, Cambridge, p. 100.

Schlossmann, H. (1938): Journal of Physiology, 92, 219. Serri, F., and Huber, W. M. (1963): In Advances in

Biology of Skin, vol. 4. The Sebaceous Glands. London, Pergamon Press, p. 1.

Shelley, H. J. (1961): British Medical Bulletin, 17, 137. Szendi, B. (1936): Archiv. fur Gynakologie, 162, 27. Villee, C. A. (1954): Cold Spring Harbor Symposia on

Quantitative Biology, 19, 186.