Paper - Histochemical observations on the germ cells of human embryos (1953)
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Mckay DG. Hertig AT. Adams EC. and Danziger S. Histochemical observations on the germ cells of human embryos. (1953) Anat. Rec.
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Histochemical Observations on the Germ Cells of Human Embryos
Donald Gr. Mckay, Arthur T. Hertig, Eleanor C. Adams and Sara Danziger
Department of Pathology, Harvard Medical School, Boston, Massachusetts and the Pathology Laboratories of the Boston Lying-in Hospital and the Free Hospital for Women, Brookline, Massachusetts
- Aided by the Institutional Grant of the American Cancer Society to Harvard Medical School and by grants from the United States Public Health Service and Department of Embryology of The Carnegie Institution of Washington.
The origin of the germ cells has been a subject of debate for a considerable period of time. The concept that they arise from the “germinal epithelium” has had many adherents including Stieve (’27), Neumann (’29) and Simkins (’28). Witschi (’48), on the other hand, in a scholarly treatment of the problem has presented sound morphologic evidence that germ cells of the human arise from the endoderm of the yolk sac or from primitive stem cells which also are the source of the endoderm. His observations indicate that the germ cells migrate from this location to the hindgut endoderm, subsequently to the mesentery of the gut and thence toward the mesonephric folds. This movement appeared to him to be aided by active movements of the individual cells associated with formation of pseudopodia and terminated with the arrival of the germ cells in the gonadal folds. He found mitoses in the migratory germ cells and noted that these cells increased in number during the migratory period.
Witschi’s observations were made on embryos measuring from 2.4 to 8mm and his findings were divergent from those of Simkins Who was unable to find “genitaloid cells” in any of the embryos of this size. Witschi wondered whether the material which Simkins dealt with was different. An alternative explanation might lie in the fact that many of the germ cells in the mesentery or gut endoderm are difficult to differentiate from the neighboring somatic cells in hematoxylin and eosin preparations and, if the material were not fixed immediately and sectioned perfectly, the cells might go unrecognized.
It is the purpose of this paper to present some histochemical studies on cells which according to the criteria of Witschi are identified as germ cells. These are characteristic large cells with prominent nuclei and in the 5mm stage (the youngest in our series) are found in the mesenchymal tissue of the mesentery of the gut, between the epithelial cells lining the gut and in the coelomic angle. They subsequently migrate to and concentrate in the genital ridge and later are localized for the most part in the cortex of the gonad. Such observations are comparable in all aspects to those made by Witschi on embryos of similar development.
Five human embryos are described ranging from 5 to 35 mm in length and of 28 to 52 days developmental age. The histochemical observations not only give insight into the metabolic activity of these germ cells but the alkaline phosphatase technique outlines them in such a striking manner that they are rendered easily Visible. With more extensive studies on a larger number of embryos, particularly those of the first 15 to 30 days of age, more accurate information on the origin of these cells may become available.
Materials and Methods
The embryos in this study were obtained at the time of hysterotomy or hysterectomy from patients at the Free Hospital for Women and the Boston Lying—in Hospital. The indications for the operations were varied and included such diseases as tuberculosis, chronic nephritis, rheumatic fever and diabetes. In spite of the presence of maternal diseases there is little reason to suspect that they caused any changes in any of the embryos examined in this group. The embryos are listed for the purpose of this report in order of size:
FHW — ”~52—1019 5 28 FHVV ~ S~5‘.Z—5005 ll 34 BLI —— S—52—2224 13 36 FHW — S—52—2716 ‘.23 42 BLI — SA52~6-10 35 52
These embryos will be described in their entirety in subsequent reports and the histories of each case will accompany them. Because of the suggested relationship between the indifferent, primitive germ cell and the so—called dysgerminoma of the ovary, an example of this neoplasm was examined by the same methods (St. Elizabeth’s Hospital, E—50e614).
The three smaller embryos were fixed in toto. The 23 mm embryo was cut into three blocks and the 35 mm fetus was cut into 6 blocks before fixation.
1. fix in ice-cold acetone at - 20 °C for 24 hours with one change.
2. Clear in oil of eedarwood overnight.
3. Transfer to xylol for one hour with one change.
4. lnfiltrate at 56-57°C. in 52-54°C. tissue mat for three hours with three changes.
5. Embed and store paraffin blocks in the deep freeze (- 20°C) until cut.
Transverse serial sections were cut at 7 u and stored as ribbons at - 20°C in the deep freeze until they were mounted. Every 25th section through the embryo was mounted for each of several histochemical reactions, i.e., sections 1, 26, 51, 76, etc. were mounted on one slide for reaction; sections 2, 27, 52, 77, etc., were mounted for another reaction, etc. When all the sections had been mounted it was possible to survey the entire length of the embryo by several different histochemical techniques. The sections were mounted 011 albumenized slides and were spread with a thin film of distilled water. The slides were then drained and dried overnight at 37°C. They were stored in the deep freeze until ready for reaction.
1. Alkaline phosphatase, (120 (lye method Manheimer and Seligman (’48)
A. Incubating mixture : Calcium alpha naphthyl phosphate 50 mg‘ Alpha naphthyl diazonium naphthalene 1,5-disulfonate 50 mg‘ Magnesium sulfate 1% 1 cc 0.1 M Barbital buffer pH 9.4 15 cc Distilled H20 84 cc
To insure solution, both powders were ground in a mortar with a small amount of water.
B. Procedure: The slides were deparaffinized in xylol, hydrated in graded acetones and rinsed brieﬂy in distilled water. They were then incubated at 6—10°C. for one hour in freshly filtered incubating mixture. After thorough washing in running tap water, they were lightly counterstained with hematoxylin and mounted in glycerin jelly. Control slides, inactivated in 1% HCl for 10 minutes before incubation, were run on each series.
Recently Fast Blue 2B was used in place of the alpha naphthyl diazonium naphthalene-1,5-disulfonate. Fast Blue 2}} gives a black rather than a brown reaction and since it does not decompose as rapidly, the reaction can be incubated at room temperature and gives no diffuse non—specific coloring of the tissue.
2. Alkaline phosphatase, ('(1rlc'ium phosphate met7m(I Pearse and Reis (’52)
The slides were reacted for three hours at 37°C. without deparaffinizing the sections, otherwise the technique was identical with that of Pearse and Reis.
3. Acitl phosphatase, azo dye method Seligman and Manheimer (’49)
A. Incubating mixture :
Sodium alpha naphthyl acid phosphate 50 mg 0.3 M acetate buffer pH 5.0 10 cc 4 M sodium chloride 90 cc Alpha naphthyl diazonium naphthalene1,5-disulfonate or Fast Blue 2B 30 mg
The slides were deparaffinized in xylol, hydrated through graded acetones, rinsed in distilled water and incubated overnight at room temperature in freshly filtered incubating mixture. After washing in running water they were mounted in glycerin jelly. No counterstain was used as it tended to detract from areas of slight reactivity. Control slides inactivated in 1% H01 for 10 minutes before incubation were run on each series. Due to the decomposition of the alpha naphthyl diazonium naphthalene-1,5-disulfonate, some ﬂaky black precipitate was formed over the slides. ‘When Fast Blue 213 was substituted, no precipitate was formed but in areas of high reactivity the reaction did not seem quite so sharply localized.
4. ZVOn—specific (as-lerase, (120 dye method Nachlas and Selig-man (’49)
A. Incubating mixture: 10 mg of alpha naphthyl acetate were dissolved in 0.5 cc acetone and added to the following solution: 2 M NaCl 50 cc 0.1 M Barbital buffer pH 7.8 20 cc 40 mg of alpha naphthyl diazonium naphthalene—1,5— disulfonate or 40 mg of Fast Blue 2B were ground in a mortar with a little distilled water and washed into the incubating mixture with 30 cc of distilled water. B. Procedure:
The slides were deparaffinized in xylol, hydrated in graded acetones, rinsed in distilled water and placed in freshly prepared and filtered incubating mixture at room temperature. At the end of 30 minutes a freshly prepared incubating mixture was filtered onto the slides and incubation was continued for anot.her 30 minutes. The slides were then washed well in running Water and mounted in glycerin jelly. No counterstain was used since it tended to detract from areas of slight reactivity. Control slides, inactivated in l‘/,. lI(_‘l for 10 minutes, were run on each series.
5. 5-]\"ueZe0tidase Pearse and Reis C52)
The slides were reacted for three hours at 38°C. without deparaffinizing the sections. The procedure was otherwise according to that of Pearse and Reis.
ln addition to these enzyme stains, alternate sections were stained for glycogen and other carbohydrate substances by the periodic acid Schiff method of McManus ( ’46). Alternate sections were also stained with eosin-methylene blue and digested with ribonuclease for the detection of ribonucleic acid. Additional sections were stained by the Prussian blue reaction for the detection of ferric iron (Bunting. ’49). VVhile acetone is not the ideal fixative for the latter stains, tests on other tissues have demonstrated no appreciable loss of glycogen, nucleic acid or iron when acetone is used. The only disagreeable feature in acetone fixation is the cellular distortion which it produces.
Description of embryos: Seliglman allralinc phosphaziasc mcthori
I. 5mm embryo (FHVV S«52—1019).
The germ cells are quite remarkably outlined by the Seligman alkaline phosphatase method. The enzyme activity appears concentrated in the cytoplasmic rim of the cells. The cell membrane is frequently quite wrinkled due to the distortion caused by acetone fixation. The nuclei do not exhibit any activity of this enzyme. The germ cells are found in this embryo in the mesenchymal tissue of the mesentery of the gut particularly concentrated just beneath and within the coelomic epithelium, between the epithelial cells lining the gut, and beneath or within the coelomic epithelium in the genital folds (figs. 1, 2). A few germ cells are in mitosis. Although this embryo was serially sectioned, all the sections were not stained by this method and no attempt has been made to account for every germ cell in this or any of the other embryos.
Fig. 1 Transverse section through 5mm human enibryo to show spinal cord, zmrtzi, urogenital ridges and primitive gut. The primitive germ cells are outlined by the Seligmun alkaline phosphatase technique and are present in the coelomic epithelium, connective tissue of the mesentery and in the developing gonadal folds. Same specimen as figure 2. (FHW S—52—l019.) X 135.
Fig. 2 Transverse section of the primitive gut, mesentery and coelomic epithelium covering the gonadal folds of 5 mm human embryo. Alkaline phosphatase is concentrated at the periphery of the cell body and does not appear in the nucleus. Same specimen as figure 1. (FHW S—52—1019.) X 300.
No germ cells were revealed in the yolk sac in this embryo by this technique.
Fig. 3 13 mm embryo. The germ (‘.8115 are most numerous in the gonadal folds but a moderate number are scattered through the connective tissue in the midline. and in the developing adrenal tiss11es which lie medial to the mesonephric. ridges. Alpha naphthyl phosphatase. (ELI S—52—2224.) Same specimen as figure 4. ><1l.2.5 before reduction.
II. 11 mm embryo (FHVV S—52—5005).
The germ cells in this embryo are present in the connective tissue of the mesentery of the gut, in gut endoderm and are most numerous in the developing genital fold. The alpha naphthyl phosphatase activity is concentrated in the cytoplasmic rim and is present in the cytoplasm of the germ cells but is not seen in the nuclei. This localization is common to the germ cells in all 5 of the embryos in this series.
III. 13 mm embryo (BLI S—52—2224).
The germ cells in this embryo are clearly delineated by the alkaline naphthyl phosphatase stain and appear concentrated in the genital ridges. A few are scattered in the connective tissue around the aorta. A relatively large number are found in the connective tissue at the root of the mesentery around
Fig. 4 13 m1n embryo. The germ cells are concentrated in the genital ridges but are also present in the mesenteric connective tissue. Alpha naphthyl alkaline phosphatase. (ELI S—52~2224.) Same specimen as figure 3. ><97.5 before reduction.
the coeliac artery nea.r the pancreas and stomach (figs. 3 and 4). None can be found in the gut endoderm in this embryo. IV. 23 mm embryo (FHW S—52—27l.6).
The gonad of this embryo has differentiated into that of a male and most of the germ cells are present in the testis, arranged in a scattered fashion along the sex cords (fig. 5). In addition, however, there are still a moderate number of germ cells in the connective tissue of the root of the mesentery (fig. 6). A few are seen in the mesentery at its point of attachment to the gut at some distance from the root of the mesentery. One germ cell is present in the gut endoderm. A few are also found in the outer regions of the adrenal cortex beneath the capsule of the adrenal and in the sympathetic ganglion tissue and in nerves near the medulla or hilus of the adrenal.
Fig. 5 The right gonad of a mm male embryo. The germ cells are scattered along the sex cords of the testis in the center of the field. On the lower left is a fragmelit of the liver, in the lower right corner, a portion of intestine, and above the gonad are several glomeruli. Alpha naphthyl phosphatase. (FHW S—52—2716.) X 97.5.
Fig. 6 23 mm male embryo. The germ cells are present in the small section of gonad in upper right corner, in the mesentery in the center and beneath the peritoneum on the left near the section of intestine. Alpha naphthyl alkaline phosphatase. The long mesentery is folded so that the gut lies in the upper left corner. (FHW S—52—2716.) X 97.5.
V. 35 mm embryo (BLI S—52—6-1L0).
The gonad of this embryo has differentiated into a11 ovary which contains the great majority of the germ cells of this specimen (fig. 7). Nevertheless, there are still a few to be found in the sympathetic ganglia and nerves at the hilus of the adrenal, and in the connective tissue of the root of the mesentery (fig. 8). A very few are seen in the mesenteric connective tissue at its point of attachment to the gut.
Fig. 7 The right gonad of a 35 mm female embryo. The ovary of this en1b1'yo contains most of the germ cells. Alpha naphthyl alkaline phosphatase. (BLI S—52—640.) X 135 before reduction.
Alkaline glycerophosphatasc method
Curiously, the germ cells cannot be distinguished as clearly with this method as with the alpha naphthyl phosphatase technique. Evidence of enzyme activity in the cytoplasmic rim of the germ cells can be found in all these embryos, but in the case of the 5 mm embryo, the surrounding mesenchyme has such a high activity that all cells are stained and the germ cells cannot be readily detected. On the other hand, the reaction of the germ cells in the ovary of the 35 mm embryo presented the same enzyme pattern and the same localization of the cells, but they were not as distinct as those stained by the alpha naphthyl technique. These differences may be explained in part by the magnesium concentration of the incubating medium, since magnesium activates alkaline phosphatase. The Seligman substrate is low i11 magnesium while the glycerophosphate substrate has a higher concentration. In the case of the 5 mm embryo, when the magnesium concentration of the alpha naphthyl substrate was raised to that of the glycerophosphate substrate, enzyme activity appeared in the connective tissue cells around the germ cells which then became less sharply demarcated. This, however, does not explain the differences found in the 35 mm embryo, which might be accounted for on the basis that the colored end product of the Seligman reaction is more intense than that of the Gromori reaction or that there is less diffusion of the end product of the Seligman technique. Another possibility is that the different substrates actually show up different enzymes.
Fig. 8 A 35 mm female embryo. A few germ cells remain in the connective tissue at the root of the mesentery although most are found in the ovary of this embryo (fig. 7). The pancreas is seen on the left and a portion of the musculature of the stomach on the right. Alpha naphthyl alkaline phosphatase. (BLI S—52— 640.) X 135.
Acid phospahatase, non-spccific cstcrasc and 5-amrwofidasc
The germ cells of these 5 embryos did not exhibit any activity of these enzymes under the conditions of fixation and preparation used.
Glycogen, iron. and ribonucleoprotein
The cytoplasm of the germ cells of the 5 mm embryo contains a visible amount of glycogen which is digested by amylase. Following digestion there is no residual material in these cells which gives the periodic acid—Schiff reaction. Small amounts of basophilic material are found beneath the cell membrane in a few germ cells. The cytoplasmic basophilia is eradicated by digestion with ribonuclease. This localiza— tion of cytoplasmic ribonucleic acid near the cell membrane has been noted i11 frog eggs by Brachet (’40, ’42) and in Arbacia eggs by Lansing and Rosenthal (232). The latter authors have adduced evidence that this surface layer of ribonucleoprotein plays a role in the transport of metabolic materials across the cell membrane. If this is true, it provides added evidence that the germ cells at these stages of development are in active metabolic exchange with their environmental tisues. VVith the Prussian blue method the germ cells exhibit no reaction.
These findings suggest that the germ cells contain small amounts of glycogen and ribonueleoprotein in their cytoplasm, but do not contain stainable amounts of periodic acid—Schitf positive substances after amylase digestion or iron at this stage of development.
The sections of the dysgerminoma were fixed and stained by the same procedures. The cells of this tumor are clearly outlined by the alpha naphthyl alkaline phosphatase reaction because the enzyme activity is concentrated in the cell membrane and is not found in the nucleus (fig. 9). The alkaline glycerophosphatase method gives a similar reaction. The other enzymes, i.e., acid phosphatase, 5—nucleotidase and nonspecific esterase are not demonstrated in these cells by the methods used, however, acid phosphatase is present in the necrotic tumor cells. Glycogen is present iii the cytoplasm and small amounts of ribonucleoprotein can be seen near the cell membrane. It was of interest to note a considerable increase in the glycogen content of cells in mitosis in this tumor.
These histochemical observations on supposed germ cells strengthen the conclusions of \Vitschi that these morphologically typical cells, arising in the caudal aspect of the yolk sac and thence coming to rest in the gonads, are indeed germ cells. Such observations, indicating probable metabolic activity further identify these cells as markedly different from those surrounding them.
fig. 9 Dysgel-minumu of adult human ovary. The individual ‘rumor cells are distinctly outlined by the brown alpha naphthyl alkaline phosphatase reaction. (St. E1izabeth’s Hospital, Boston 15-50-614, courtesy D1‘. James Grzlham.) X 300.
Pathologists have long considered that the dysgerminoma of the ovary and its morphologic twin, the seminoma of the testis, accurately recapitulate the morphogenesis of the primitive gonad. That such interpretation of the nature of these embryonal carci11omata is correct is indicated by the histochemical similarity of the tumor cells and the primordial germ cells of the early embryo.
- Alkaline and acid phosphatase, 5—nucleotidase, non~specific esterase, glycogen, periodic acid-Schiff and ribonucleo— protein staining methods have been used on 5 embryos ranging from 5 to 35 mm in length and from 28 to 52 days ovulation age.
- The germ cells of these embryos exhibit a high alkaline phosphatase activity (Seligman technique) in the cytoplasmic rim which microscopically isolates these cells very sharply from the surrounding tissues.
- Germ cells were found in the same locations as those described by Witschi. In addition they were found in the root of the mesentery, in the adrenal and in the sympathetic ganglion and nerve tissue at the hilus or medulla of the adrenal in the 23 mm and 35 mm embryos.
- The histochemical reactions of the cells of one dysgerminoma were identical with those of the germ cells, which tends to confirm the idea that the latter are the cells of the origin of dysgerminomas.
- The presence of a high alkaline phosphatase activity at the germ cell membrane suggests that these cells are in a state of active metabolic interchange with their surrounding tissues at these stages of development.
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