THE ANATOMICAL RECORD 235:604-610 (1993) Isolation of Chick Primordial Germ Cells From Stages 4-8 Embryos GEORGE MATSUMURA AND MARJORIE A. ENGLAND Department of Anatomy No. 2, Hokkaido University, Sapporo 060, Japan (G.M.); Department of Anatomy, University of Leicester, Leicester, Great Britain (M.A.E.) ABSTRACT Chick embryo primordial germ cells (PGCs) stages 4 4 were manually isolated for the first time from the late hypoblast layer. They were confirmed to be PGCs by periodic acid-Schiff (PAS) staining and examination by scanning electron microscopy (SEM). They were subsequently introduced onto a variety of artificial substrata. On two dimensional substrata, the cells change from a spherical shape covered with numerous microvilli to a rounded cell with a “skirt” of cytoplasm. Eventually a process projects from one side of the smooth cell. On a three dimensional substrate the PGCs change from a spherical shape covered with numerous microvilli to a smooth surfaced cell with a long single process. It is concluded that the PGCs which are originally spherical in situ in stage 4 alter their morphology both in vivo during their migration and in vitro studies. o 1993 Wiley-Liss, Inc. MATERIALS AND METHODS Primordial germ cells (PGCs) in the early chick embryo have been extensively studied since 1870 Fertilized White Leghorn chicken eggs were incu(Waldeyer, 1870). The focus of numerous studies has bated at 37°C until stages 4-8 (Hamburger and Hamilbeen the origin, location, and migration of these cells ton, 1951). The embryos were then mounted as for New into the gonads (Swift, 1914; Goldsmith, 1928; Rogul- Culture (New, 1955) and all excess yolk particles ska, 1968; Fujimoto et al., 1976a, b; Fargeix et al., washed off. 1980; Eyal-Giladi et al., 1981; England, 1983; England et al., 1986; Kuwana et al., 1986, 1987; Nakamura et PGCs in Normal Stage 4 Embryos al., 1991; Ukeshima et al., 1991). The PGCs originate Embryos mounted by New Culture were fixed in in the epiblast (Eyal-Giladi et al., 19811, translocate to Karnovsky’s fluid (Karnovsky, 1965) for 3 hours and the hypoblast, and finally during the primitive streak and headfold stages separate from the hypoblast. They transferred to cacodylate buffer (Plumel, 1948). The migrate along a fibrous band (Wakely and England, ectoblast layer was dissected off over the anterior area 1979) which is rich in fibronectin, sulphated glycosami- pellucida/area opaca border to expose PGCs within the noglycans (Critchley e t al., 1979), and collagen type I underlying hypoblast layer. The specimens were then (England et al., 1982). This band forms a n arc located prepared for scanning electron microscopy (SEM). a t the cephalic border of the area pellucidalarea opaca Isolation of PGCs for Culture and its position exactly corresponds with the location of The New Culture glass ring was filled with saline. the PGCs described by many investigators (Swift, 1914; England, 1983; Kuwana et al., 1987). As the The PGCs are known to populate a n area along the PGCs move along this fibrous band they physically de- cephalic area pellucida/area opaca border. This region form and rearrange the fibers (England, 1983; England was identified (Fig. 1)and the hypoblast overlying this et al., 1986). Subsequently, the PGCs enter the devel- border was carefully dissected away in a single piece oping vascular supply (Dubois, 1969) and ultimately using Rebutia hybrid cactus needles mounted on wooden cocktail sticks (England, 1981). As the hypopopulate the gonads (Ukeshima et al., 1991). Although the PGCs are large, round, periodic acid- blast was separated up to 30-40 PGCs floated free and Schiff (PAS)-positive cells they have not been isolated came to settle on the underlying ectoderm layer. In and cultured for in vitro studies in the early chick em- addition to PGCs separating, yolk granules also float bryo. Fujimoto et al. (1976a) isolated chick PGCs from free and come to lie with the PGCs. The only distinthe circulating blood a t stage 8 and cultured them in guishing features we were able to recognize between vitro for a short time (unspecified) to study their mode of migration and morphology. To date these have been the youngest chick PGCs isolated for in vitro culture. In the present study, we report the isolation, culture, Received April 1, 1992; accepted September 11, 1992. and examination of PGCs of the much earlier stages Address reprint requests to Dr. G. Matsurnura, Department of 4-8 in the chick embryo. Anatomy No. 2, Hokkaido University, Sapporo 060, Japan. 0 1993 WILEY-LISS, INC. ISOLATION OF PRIMORDIAL GERM CELLS 0 /. 0 .\* foo Fig. 1. A diagram to illustrate the position of the chick PGCs at stages 4-8. the yolk and the living PGCs were a n elliptical shadow on the PGCs and their size. Preparation of Substrates for PGCs PGCs were removed for in vitro studies using fine glass pipettes attached to a mouthpiece and rubber tubing. They were transferred to a variety of substrata and media. All of the equipment used was sterilized by overnight ultraviolet radiation. The substrata included glass coverslips, plastic (Thermonox Nunc., Inc.) coverslips, Sterispon (Allen & Hambury Ltd., London), and Spongostan (Ferrosan, Denmark). Sterilized coverslips (Thermonox) were used as supplied by the manufacturer. Glass coverslips were washed in distilled water, some were further washed in acid alcohol (0.5%HC1 in 70% ethanol) and then dried in a plastic Petri dish and placed overnight in the ultraviolet radiation cabinet. The media used included normal physiological saline and Medium 199 (Gibco). In addition to using plain glass and plastic surfaces, these same substrata were coated with purified collagen or 1% agar or rat tail collagen. These conditions provide a direct comparison with the work described by Kuwana et al. [1986,1987]. Purified collagen as supplied by the manufacturer, or diluted, was placed on the coverslip the day before use and allowed to dry overnight in a covered Petri dish a t room temperature. This produced either a thick or a thin layer of collagen on the coverslip. These coated coverslips were used a s follows: a drop of medium was placed on the coverslip and the PGCs added immediately. They were either left at room temperature for 0 minutes, 15 minutes, and 30 minutes before fixation or incubated at 37°C for 30 minutes before fixation in Karnovsky’s fluid. Alternatively the coated coverslips were dried overnight and the next day the entire coverslip was added to either saline or Medium 199 (Gibco) and left overnight a t room temperature. The PGCs were added to the coverslip and incubated for 15 and 30 minutes at room temperature. They were then fixed in Karnovsky’s fluid for SEM. A solution of rat tail collagen (0.4%)was prepared by adding distilled water to a bottle of collagen and shak- 605 ing it until the collagen dissolved. It was stored at 4°C in the refrigerator. Before use the collagen was allowed to warm to room temperature. Then it was added to the coverslip and either left a s a drop or spread across the coverslip. Coverslips were dried overnight at room temperature in a Petri dish. The PGCs were then added for 0 hours, 3 minutes, 15 minutes, 30 minutes, and 40 minutes. Rat tail collagen with acetic acid (Kuwana et al., 1987) was also used by adding 1 mg of collagen/ml of 1% acetic acid. Either a single drop was used or spread across the coverslip and this was dried overnight in a Petri dish at room temperature. These coverslips were then used as the coverslips above. Another substrate was prepared by dissolving agar in distilled water to make a 1% solution (at 60°C) and then using a warmed pipette placing a drop of agar on the coverslip. After cooling, PGCs in saline were placed on the agar and incubated for 15 minutes a t room temperature. They were then fixed in Karnovsky’s fluid. The stages 4-8 PGCs were removed from the embryos and placed in contact with one of the above substrates and left for either 0, 10-15, or 30 minutes a t room temperature. They were subsequently prepared for SEM by carefully introducing drops of Karnovsky’s fluid (Karnovsky, 1965) onto the medium used. Comparisons were also made with yolk granules. Sterispon and Spongostan Sterispon and Spongostan were used a s supplied by their manufacturers. A very thin slice of either was cut using a razor blade. The thin slice of gelatin was attached to a Cambridge stub using double-sided Scotch tape (3M). PGC preparations were added directly to the gelatin and left in contact for 15 and 30 minutes and prepared for SEM. Scanning Electron Microscopy Specimens were fixed for 3 hours and then in buffered cacodylate (Plumel, 1948) overnight. The PGCs were postfixed in 1%0, 0, in 0.1 M cacodylate buffer for 30 minutes and dehydrated in a n ascending series of ethanol/water from 30%-100%. They were transferred to 100% acetone and stored in sealed vials. The specimens were critical point dried in a Polaron critical point drier by acetone replacement. They were then mounted on Cambridge stubs using silver Dag and coated with 20 nm of gold in a Polaron coating unit. The PGCs and their substrata were viewed in a DS130 International Scientific Instruments scanning electron microscope a t a n operating voltage of 9-15 KV. Photographs were taken using a fine grain ultraslow film (Kodak Technical Pan 2415). Periodic Acid-Schiff Staining Isolated PGCs and wholemount chick embryos were also stained with PAS stain to determine the position and appearance of PGCs in vivo. Isolated PGCs were also stained with PAS to prove they were PGCs and not somatic cells. Both the wholemount embryos and the isolated PGCs were fixed in Karnovsky’s fluid for 3 hours, buffered in 0.1 M cacodylate overnight. They were then stained in a 1% aqueous periodic acid solution (BDH Poole) for 20 minutes. They were transferred to Schiff‘s solution for 30 minutes, rinsed in dis- 606 G. MATSUMURA AND M.A. ENGLAND Fig. 3. An isolated PGC. Note the nucleus (n). x 750 to their substrate often exhibit after 15-20 minutes a sheet of cytoplasm projecting from the cell which conFig. 2. Chick PGCs in vivo in the stage 4 embryo as viewed by scanning electron microscopy. The surrounding hypoblast cells (h) tacts the substrate (Fig. 6). This sheet resembles a remain in contact with the PGCs. Note the presence of microridges (r) “skirt” projecting in a complete circle from the cell and short microvilli (v). x 3,125. base. Numerous microvilli are present on this skirt. The upper regions of the cells retain their spherical shape and microvilli. Some blebs also appear on the tilled water three times, mounted on a slide in water, upper surface. PGCs placed in contact with thick layers of collagen and the coverslip sealed with nail varnish. These were compared with living PGCs mounted on a glass slide in initially exhibit a similar appearance to those cells distilled water without staining. Photographs were placed on glass or plastic coverslips. After 15-30 mintaken immediately on Kodak Ektachrome EPY135 or utes blebs are present (Fig. 7) and the cells appear to be deforming the surrounding fibers of collagen. on Kodak Technical Pan 2415. The PGCs adhere best to the gelatin substrate SterRESULTS ispon or Spongostan. Although 30-50 PGCs were PGCs In Situ and PAS Staining placed on a single coverslip (or substrate) often only 10 PGCs in situ in the hypoblast layer of the stage 4 PGCs or less adhered and were subsequently examined chick embryo have a characteristically spherical shape. by SEM. When 30-50 PGCs were introduced on the By SEM their surfaces are covered with numerous three dimensional gelatin latticework, approximately small microridges and some punctate microvilli (Fig. the same numbers were examined by SEM. At zero minutes, the PGCs resemble those cells in situ and 2). In those wholemount stage 4 embryos stained by those cells introduced on other substrata (Fig. 8). By PAS a large number of PGCs were evident in the re- 15-30 minutes, however, the cells often exhibit a broad gion anterior to the primitive streak and concentrated process which varies from specimen to specimen in its in the region of the area opacatarea pellucida border. length and breadth (Figs. 9,101. The spherical shape of PGCs isolated from these specimens or PAS stained the PGC has altered to a more flattened corpus with a following isolation confirmed that these large cells are broad projecting process. Few or no microvilli are PGCs. By light microscopy, the cells are large and present on the upper surface of the germ cell. By 40 rounded with numerous refractile lipids in their cyto- minutes some of the processes are longer than those plasm and a n identifiable nucleus (Fig. 3). They are present a t 15-30 minutes. PAS positive. They often have a blunt process evident DISCUSSION bulging from one side of the cell. PGCs and Artificial Substrata With the exception of the two gelatin substrata, Sterispon and Spongostan, few PGCs adhere to the combinations of substrata employed in this study. The findings are summarized in Table 1. Those stages 4-8 PGCs placed on glass coverslips or Thermonox plastic initially resemble those PGCs observed in situ (Fig. 4). They are spherical but covered with numerous long microvilli and punctate blebs. Only a small number of microridges are present. There are additionally, however, within 15 minutes some small blebs present (Fig. 5). Those cells which adhere /solation of PGCs In the present study, stages 4-8 living chick PGCs were isolated for the first time from the late hypoblast layer. It should be emphasized that this method is only achieved with lengthy experience. The average size of chick PGC is 11.4 pm (Kuwana et al., 1987). As they are in a mixed population of yolk and hypoblast cells their separation by hand is difficult. The two criteria used for separation were size and a n elliptical shadow on the PGC edge. The isolated cells were proven to be PGCs by PAS staining and examination by SEM. The cells were subsequently introduced onto a variety of artificial sub- 607 ISOLATION OF PRIMORDIAL GERM CELLS TABLE 1. A summary of combinations of in vitro substrata tried in culturing stages 4-8 PGCs' Substrate used Plain glass coverslip Thermonox plastic coverslip Glass coverslip plus 1%agar Glass coverslip plus purified collagen Glass coverslip plus rat tail collagen Thermonox plus 1% agar Thermonox plus purified collagen Thermonox plus rat tail collagen Sterispon Spongostan Stages 4-8 PGCs + Saline __ \ Stages 4-8 PGCs + Medium 199 __ \ \ \ \ \r L \+ \+ I+ \+ '--\, no attachment; - \, majority of specimens not attached; ,, approximately half specimens attached; ,+ , approximately three-quarters of all specimens attached. strata. On glass, plastic, or thin collagen layers, the cells change from a spherical shape covered with numerous microvilli to a rounded cell with a skirt of cytoplasm. Eventually a process projects from one side of the cell which loses its microvilli. On thick collagen, or three dimensional gelatin lattices, the PGCs project broad processes and lose their microvilli. Cell Movement Some features of the present study closely resemble the morphological stages of attachment on glass, plastic, and collagen as described by Kuwana et al. (1987). There are, however, several major differences between the present work and the study by these authors. They describe the PGCs isolated from the circulating blood somite stage 19 in vitro as migrating on a thin collagen layer either in isolation or in conjunction with gonadal ridge tissue. Initially the cells are covered by numerous microvilli which adhere only with the tips of their microvilli. If no gonadal ridge tissue was present, the PGC elongated on its substrate without movement. If gonadal ridge tissue was present, the PGC contacted the substrate with a cytoplasmic skirt. The cell then extended a long pseudopodium which swelled gradually. Eventually the main cell body moved through the pseudopodium toward the gonadal ridge. In the present report, the initial attachment and migration stage are similar to that reported by Kuwana et al. (1987). However our PGCs were never placed in the vicinity of gonadal tissue, but they assumed the morphology of those cells moving toward ridge tissue. We never ob- Fig. 4.A stage 5 PGC isolated on a glass coverslip and viewed by scanning electron microscopy. x 3,072. Fig. 5.A scanning electron micrograph of a stage 5 PGC cultured on a glass coverslip for 30 minutes. Often by 15-30 minutes some small blebs (b) are present. x 3,658. Fig.6.A scanning electron micrograph of a stage 4 PGC cultured on Thermonox plastic and rat tail collagen for 15 minutes. Note the sheet of cytoplasm on the substrate (arrowheads). x 4,225. 608 G. MATSUMURA AND M.A. ENGLAND Fig. 7. A stage 8 PGC incubated in Medium 199 for 30 minutes at 37°C on rat tail collagen. Note the blebs (b). X 7,310. served the flattened morphology described for PGCs not exposed to gonadal tissue. These reported differences may be due to the very different ages of the PGCs used in the two studies. Kuwana et al. (1987) isolated 19 somite stage embryo PGCs, while ours were from the stages 4-8. It would not be surprising to discover there are some cell changes between the pre- and early migratory stages 4-8 and the late migratory 19 somite stage PGCs. In fact, while some of their features are known to remain stable, others change dramatically over this period. Fujimoto et al. (197613) demonstrated that lipids (as stained with Oil red 0 or Sudan black B) remain even during late stages of development. They also demonstrated that PAS positive glycogen is present in the cytoplasm of PGCs from stage 4- day 5. Changes which occur have also been reported. Swartz and Domm (1972) found that PGCs undergo mitotic division during migration and the initial population of PGCs is therefore not necessarily the same population migrating in the vascular network. Fujimoto et al. (1976a) also reported that yolk granules are almost completely dissipated by the time the PGCs reach the germinal ridge. In the mouse embryo, changes have been reported between migratory and post-migratory PGCs in vitro (Donovan et al., 1986). Migratory cells were shown to be motile in vitro and demonstrated characteristically invasive behavior. Gonadal PGCs were no longer invasive and showed little locomotory activity. Similar changes could be occurring between the pre- and early migratory PGCs and the much older migrating PGCs. Our method of isolation is dependent upon the chick PGCs separating naturally from the late hypoblast layer. These PGCs could be described as at the early end of their migratory phase. Their next phase of development includes actively migrating along a fibrous substrate rich in fibronectin (England, 1983). The morphology of the PGCs changes dramatically a s the spherical cells project long pseudopodia and move toward and along this substrate. The newly isolated stage 4 PGC movements are reminiscent of those described on a cellular substrate (England, 1983) rich in fibronectin. Fig. 8. A stage 5 PGC isolated and introduced onto Sterispon for 10 minutes. x 5,814. Culturing Techniques The culture of stages 4-8 chick PGCs using the routine laboratory substrata and culture media is achieved only with difficulty. With the exception of the two gelatin lattices, Sterispon and Spongostan, the other substrata used were clearly failures in this study (Table 1). This failure rate is similar to attempts by other authors to culture PGCs in other species. DeFelici and McLaren (1983) found the mouse PGCs to be consistently nonadherent to glass, plastic, or gelatin coated substrata. Greater success in the survival of proliferating mouse PGCs has been achieved with the introduction of a cellular substrate (Donovan e t al., 1986) and growth factors (MGF-SCF). The leukemia inhibitory factor (LIF) in conjunction with feeder layers of an established Sertoli cell line TM, (De Felici and Dolci, 1991) has recently been shown to sustain the survival of mouse PGCs from 10.5 dpc embryos for at least 3 days. Sterispon and Spongostan are routinely used in surgical procedures and act as scaffolding for tissue reconstruction. Their advantage in tissue culture systems is the provision of a three dimensional substrate for cell movement and architecture similar to the normal morphology encountered in vivo. It is known that the cell morphology alters between two and three dimensional substrata (England and Wakley, 1979) and the changes in this report would confirm this observation. The morphology of the cells on glass, plastic, and collagen is different from that on the gelatin lattices. The morphology of the PGCs on the two dimensional substrata closely resembles that of the migrating PGCs described in the early primitive streak stage chick embryo (England, 1983). Their shape changes on the gelatin lattices most closely resemble the changes observed in amphibian PGCs introduced and cultured on chick embryos (England et al., 1986). This variation in appearance of the same aged PGCs is probably also a n indication that the cell spreading behavior is influenced by 609 ISOLATION OF PRIMORDIAL GERM CELLS LITERATURE CITED Critchley, D.R., M.A. England, J. Wakely, and R.O. Hynes 1979 Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature, 280:498-500. De Felici, M., and S. Dolci 1991 Leukemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM, feeder layers. Dev. Biol., 147:281-284. DeFelici, M., and A. McLaren 1983 In vitro culture of mouse primordial germ cells. Exp. Cell Res., 144t417-427. Donovan, P.J., D. Stott, L.A. Cairns, J . Heasman, and C.C. Wylie 1986 Migratory and postmigratory mouse primordial germ cells behave differently in culture. Cell, 44:831-838. Dubois, R. 1969 Le mecanisme d' entree des cellules germinales primordiales dans le reseau vasculaire, chez l'embryon de Poulet. J. Embryol. Exp. Morphol. 21:255-270. England, M.A. 1981 Application of the SEM to the analysis of morphogenetic events. J . Microsc 123:133-146. England, M.A. 1983 The migration of primordial germ cells in avian embryos. In: Current Problems in Germ Cell Differentiation. A. McLaren and C.C. Wylie, eds. Symposium of British Society for Developmental Biology, Cambridge University Press, Cambridge, pp. 91-114. England, M.A.. D.R. Critchlev, and G. Shellswell 1982 Collagen Type .. -1 in the early chick embryo. J . Anat. 134:626-628. England, M.A., and G. Matsumura 1992a The localisation of primitive streak stage chick primordial germ cells. J. Anat., 180:374. England, M.A., and G. Matsumura 199213 Primordial germ cells in the primitive streak stages chick embryo as studied by scanning electron microscopy. J . Anat., submitted. England, M.A., A.P. Swan, and P. Dane 1986 The migration of amphibian primordial germ cells in the chick embryo. Scanning Electron Microsc., 111:1175-1182. England, M.A., and J. Wakely 1979 Evidence for changes in cell shape from a 2-dimensional to a 3-dimensional substrate. Experientia, 35t664-665. Eyal-Giladi, H., M. Ginsburg, and A. Farbarov 1981 Avian primordial germ cells are of epiblastic origin. J . Embryol. Exp. Morphol., 65:139-147. Fargeix, N., E. Didier, J. Guillat, and M. Damez 1980 Utilisation de diverses lectives fluorescentes dans l'etude des cellules germinales en migration chez l'embryon d'Oiseau. C.R. Acad. Sci. Paris, t. 290 Serie D. 999-1002. Fisher, M., and M. Solursh 1979 The influence of the substratum of mesenchyme spreading in vitro. Exp. Cell Res. 123:l-14. Fujimoto, T., T. Ninomiya, and A. Ukeshima 1976a Observations of the primordial germ cells in blood samples from the chick embryo. Dev. Biol., 49:278-282. Fujimoto, T., A. Ukeshima, and R. Kiyofuji 1976b The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat. Rec., 185:139-154. Goldsmith, J.B. 1928 The history of the germ cells in the domestic fowl. J. Morphol. Physiol., 46:275-315. Hamburger, V., and H.L. Hamilton 1951 A series of normal stages in the development of the chick. J . Embryol. Exp. Morphol., 88: 49-92. Karnovsky, M.J. 1965 A formaldehyde-gluteraldehyde fixative of high osmolarity for use in electron microscopy. J . Cell. Biol., 27: 137a. Kuwana, T., H. Maeda-Suga, and T. Fujimoto 1986 Attraction of chick primordial germ cells by gonadal anlage in vitro. Anat. Rec., 215:403-406. Kuwana, T., Y. Miyayama, Y. Kajiwara, and T. Fujimoto 1987 Behaviour of chick primordial germ cells moving toward gonadal primordium in vitro: Scanning electron microscopic study. Anat. Rec., 219:164-170. Matsumura, G., and M.A. England 1992 The isolation of chick primordial germ cells at the primitive streak stages. J. Anat., 180: 374. Nakamura, M., T. Kuwana, Y . Miyayama, K. Yoshinaga, and T. Fujimoto 1991 Ectopic colonization of primordial germ cells in the chick embryo lacking the gonads. Anat. Rec., 229:109-115. New, D.A.T. 1955 A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol., 3:326-331. Plumel, M. 1948 Tampon a u cacodylate de sodium. Bull. SOC.Chim. Biol. (Paris) 30:129-130. Rogulska, T. 1968 Primordial germ cells in normal and transected duck blastoderms. J. Embryol. Exp. Morphol., 2Ot247-260. Swartz, W.J., and L.V. Domm 1972 A study on division of primordial germ cells in the early chick embryo. Am. J . Anat., 135t51-70. I Fig. 9. A stage 5 PGC isolated and introduced onto Sterispon for 30 minutes. Note the cellular process (arrowhead). x 5,172. Fig. 10. A stage 5 PGC introduced onto Sterispon for 30 minutes. Compare this Figure with Figure 8. X 2,610. the substratum (Fisher and Solursh, 1979). Interestingly, the chick PGCs populating the gonads lost their morphology of migration and become rounded (Ukeshima e t al., 1991). They are similar in appearance to premigratory PGCs (England and Matsumura, 1992a,b; Matsumura and England, 1992). ACKNOWLEDGMENTS The authors are grateful to Miss D. Meecham for printing the scanning electron micrographs produced by Mr G.L.C. McTurk of the Leicester Scanning Electron Microscope Unit. We are also pleased to thank Mrs. L. Bradshaw for excellent secretarial services. Dr. Matsumura is very grateful to the Wellcome Trust for the travel grant awarded to him to support these studies. 610 G. MATSUMURA AND M.A. ENGLAND Swift, C.D. 1914 Origin and early history of the primordial germ cells in the chick. Am. J. Anat., 15:483-516. Ukeshima, A,, K. Yoshinaga, and T. Fujimoto 1991 Scanning and transmission electron microscopic observations of chick primordial germ cells with special reference to the extravasation in their migration course. J. Electron. Microsc., 40:124-128. Wakely, J., and M.A. England 1979 Scanning electron microscopical and histochemical study of the structure and function of basement membranes in the early chick embryo. Proc. R. SOC.Lond. (B). 206:329-352. Waldeyer, W. 1870 Eierstock und Ei. Els Beitrag zur anatomie und entwicklungs geschichte der sexualorgane. W. Engelmann Leipzig.