/. Embryol. exp. Morph. Vol. 56, pp. 239-252, 1980 239 Printed in Great Britain © Company of Biologists Limited 1980 Origin and cytochemistry of the animal dimple granules in Discoglossus pictus (Anura) eggs By PIERO ANDREUCCETT11 AND CHIARA CAMPANELLA1 From the Institute of Histology and Embryology, University of Naples, Electron Microscopy Center, Faculty of Sciences, Italy SUMMARY At the centre of the animal hemisphere of the 1000 /*m ovarian oocytes of Discoglossus pictus there is the germinative area, a slightly depressed disc, 700 /tm wide. During the passage of the oocyte through the oviduct this area becomes invaginated to form the 'animal dimple', which is the only site where successful sperm entrance can occur. Granules (G) with a central electron-dense core and a peripheral portion containing sparse fibrous material are found in the peripheral cytoplasm of the animal dimple. The origin and the cytochemistry of G's have been investigated. G's originate from Golgi complexes located in the peripheral cytoplasm of the germinative area only. Recently formed G's migrate to the oocyte cortical region, where vesicles appear to participate in G's matura- tion. The core of G's can be digested by pronase, is negative to a test for acid phosphatase detection, and contains a few polysaccharide complexes. In view of their origin from Golgi complex, their location in the oocytes and their exocytosis upon activation, G's seem to correspond to the typical cortical granules of Anurans. However, they appear to have a macromolecular content different from that of the cortical granules. INTRODUCTION It is well known that cortical granules are typical components of a large majority of eggs from several phyla. Among Amphibians, Anuran eggs have cortical granules which measure 2-0 fim, contain a granular matrix of homogeneous aspect and are distributed almost in a single layer in the whole cortex (Wischnitzer, 1966; Grey, Wolf & Hedrick, 1974). During oogenesis they originate from Golgi complexes in 250-350 /on oocytes (Balinsky & Devis, 1963; Ward & Ward, 1968) at the whole oocyte periphery (Favard & Favard Sereno, 1969). Their mucopoly- saccharide content is released at fertilization in the perivitelline space (Balinsky, 1966; Kemp & Istock, 1967; Grey et al. 1974) and in Xenopus laevis they participate in the formation of the F-layer (Wyrick, Nishihara & Hedrich, 1974). The egg of Discoglossus pictus is exceptional among Anurans since it does not contain typical cortical granules (Campanella, 1975). However, in the animal 1 Authors' address: Istituto di Istologia ed Embriologia, via Mezzocannone n.8, 80134 Napoli, Italia. 16-2 240 P. ANDREUCCETTI AND C. CAMPANELLA dimple, a restricted region of the animal hemisphere, some granules (G's) are present, which might be analogous to cortical granules, since they participate in the activation process. G's, however, differ from typical Anurans cortical granules in the following characteristics: (1) they measure about 0-5jam; (2) they have an irregular shape and a crystalline core; (3) they are found only in the dimple, aligned between filament bundles and intermingled with numerous vesicles, for a depth of about 10 [im (Campanella, 1975). The dimple is a funnel-shaped region where finger-shaped microvilli and filaments - as well as granules - are found in contrast to the rest of the egg periphery. Moreover, the most relevant feature of the dimple is that it is the only site where spermatozoon entrance is successful (Hibbard, 1928). The dimple is formed in the ovarian oocyte germinative area (Hibbard & Wintrebert, 1928). The germinative area is a slightly depressed disc which surrounds and includes the animal pole and appears in the oocyte at the end of vitellogenesis. After ovulation the germinative area further invaginates and gives rise to the dimple (Denis-Donini & Campanella, 1977). The purpose of this paper was to study the origin and cytochemistry of the dimple granules in an effort to elucidate the nature of these organelles and to understand to what extent they correspond to typical Anuran cortical granules. A question of particular relevance is whether G's originate directly in a restricted area such as the germinative area or are first produced at the whole egg periphery and subsequently concentrated in the germinative area. The results show that C's originate from the Golgi complexes located only in the germinative area. The G's central core is predominantly protein but also contains small amounts of polysaccharide complexes. No acid phosphatase is found in G's. MATERIALS AND METHODS Adult Discoglossus pictus (painted frog) females were captured in the neigh- bourhood of Palermo (Italy) in February-May. Their ovaries were excised and various oogenetic stages were selected. Oocytes were fixed in Smith's fixative and celloidin-paraffin embedded for light microscopy; they were stained with PAS for glycoprotein detection. Both oocytes and uterine eggs were fixed for electron microscopy in 2-5-3-0 per cent (w/v) glutaraldehyde in 0-067 phosphate buffer at 7-4 pH. They were then postfixed in 2 % (w/v) osmium tetroxide in the same phosphate buffer, dehydrated and embedded in Araldite-Epon. Ultra- thin sections were stained with uranyl acetate followed by lead citrate (Reynolds, 1963) and observed with a Siemens Elmiskop 1 A. Origin and cytochemistry of the animal dimple granules 241 Cytochemistry Proteins. In order to test for the presence of proteins in G's, ultrathin sections, collected on copper grids, were treated with 15% (v/v) H2O2 (110 vol.) for 30min at 27 °C, rinsed in distilled water and incubated in 0-1-0-5% (w/v) pronase (Calbiochem) in 0-01 M phosphate buffer for 40 min at 37 °C (Anderson & Andre, 1968; Hubert, 1974). Controls were incubated in phosphate buffer, without pronase. Acid phosphatase. To examine the possible lysosomal nature of G's, acid phosphatase histochemistry was performed. Oocytes, uterine eggs and liver, as control tissues, were fixed in 4% (w/v) glutaraldehyde in 0-1 M sodium cacodylate buffer at pH 7-4 for 5 h at 4 °C. During fixation, the samples were cut into 0-5 mm square pieces. They were then rinsed several times in cacodylate buffer to which 7 % (w/v) sucrose was added and incubated at room temperature for 90 min in the Gomori medium, modified according to Barka & Anderson (1962). The samples were rinsed in the same cacodylate buffer, postfixed for 1 h in 1 % (w/v) osmium tetroxide in veronal-HCl buffer at pH 7-4, dehydrated in acetone and embedded in Araldite-Epon (Anderson, 1968). Controls were incubated in the Gomori medium without /?-glycerophosphate or in a com- plete medium containing 0-01 M-NaF as an inhibitor (Brunk & Ericsson, 1973). Polysaccharide complexes (a) Phosphotungstic acid (PTA). Floating sections were exposed to 1 % (w/v) PTA at room temperature for 5 min, according to Marinozzi (1968). (b) Periodic acid-thiocarbohydrazide (TCH)-silver protein (AgP) (Thiery, 1967). Silver grey ultrathin sections were incubated by floating in the following media: (a) 1% (w/v) periodic acid in distilled water for 40 min; (b) 0-2% TCH (w/v) in diluted acetic acid for 24, 48 or 72 h; (c) 1% (w/v) AgP in distilled water for 30 min in the dark. Each step was followed by careful and repeated rinsing in distilled water. Control sections were not exposed to periodic acid or TCH. RESULTS (A) The origin of G's Observations performed on several oogenetic stages show that G's are present only in late stages of oogenesis, during further development of the germinative area. The germinative area appears in oocytes about 900-1000 [im in diameter, in which the nucleus has recently migrated towards the animal pole. It is a characteristically lightly pigmented disc 700 [im in diameter, which can be readily distinguished from the rest of the heavily pigmented animal hemisphere both by its colour and because it is slightly depressed with respect to the surface of the oocyte (Denis-Donini & Campanella, 1977). Along the germinative area surface and the vitelline envelope a conspicuous perivitelline space is found 242 P. ANDREUCCETTI AND C. CAMPANELLA Fig. 1. Paraffin section of the animal hemisphere of a 900 /*m oocyte. The nucleus (N) is nearby the animal pole. The region limited by the arrowheads is the germina- tive area (GA). The germinative area disc is slightly depressed and filled with material strongly positive to PAS (large arrow), x 320. containing PAS-positive material (Figs 1, 2). At the germinative area long microvilli and macrovilli project into the perivitelline space (Fig. 2). On the rest of the oocyte surface, the vitelline envelope is in close contact with the plasma membrane and both microvilli and macrovilli are embedded in the envelope; G's are absent (Fig. 3). The peripheral cytoplasm of the germinative area can be divided in two layers: ZA and ZB. ZA extends for about 25/tm from the oocyte surface and contains glycogen, mitochondria, few pigment granules, vesicles and multi- vesicular bodies (Fig. 2). Yolk platelets and pigment granules characterize the underlying zone ZB, which extends to the perinuclear area and contains glycogen and mitochondria as well (Fig. 2). Mature G's are found in ZA cortical cyto- plasm, where they are arranged in more than a single layer (Figs 2, 4). G's contain one or two centrally located electron-dense cores surrounded by a less electron-dense material in which fibrils and granules are observed (Fig. 4). These constituents are bound by an even apposition of dense material which is separated from G's by a cleft of about 20 nm (Fig. 4, inset). Golgi complexes are found in ZA as well as in ZB. At their maturing face a large number of vesicles of varying size and contents are found (Figs 5, 6).