Origin and Cytochemistry of the Animal Dimple Granules in Discoglossus Pictus (Anura) Eggs

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/. 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 germinative 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 multivesicular 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 cytoplasm, 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). By contrast, the forming face is characterized by the presence of small patches of electron-dense material as well as of vesicles. In Fig. 5 the terminal ends of Golgi saccules are dilated. In the vicinity of the saccules there is a large number of smooth surfaced vesicles of varying diameters. Some of these are probably derived from the saccules by pinching off, as they have electron density and size similar to the saccule terminal ends. Other vesicles appear as secretory Origin and cytochemistry of the animal dimple granules 243

Fig. 2. Ultrathin section of a portion of the germinative area of a 1000 /tm oocyte. in (he vitelline envelope (VE) one can observe, projecting from the follicular cells (FC), macrovilli (MA). Macrovilli are also seen in the fibrous perivitelline space content (PSC), which is located between the vitelline envelope and the plasma membrane. Also the microvilli (Ml) are partially embedded in the perivitelline space content. Granules (G) are present at the peripheral zone A(ZA) where multivesicular bodies (MVB) are also found. In the subjacent cytoplasm, zone B(ZB), Golgi complexes (GC) pigment granules (P), yolk platelets (Y) are present. N = nucleus; GLY = glycogen islets; M = mitochondria clusters, x 3800. Inset: a macrovillus containing microtubules (small arrows) x 2900 244 P. ANDREUCCETTI AND C. CAMPANELLA

Fig. 3. Vegetative hemisphere of a 1000/*m oocyte. Macrovilli (MA) and microvilli (MI) interdigidate in the vitelline envelope (VE). Both perivitelline space content and G's are absent. Arrow = endosomes; PYP = primordial yolk platelets; FC = follicular cell; GLY = glycogen. x 18000. vacuoles due to their flocculent contents and their confluence with small vesicles. In Fig. 6 the secretory vacuoles next to Golgi saccules contain a rather flocculent material, which may be a precursor of G contents. In addition to the flocculent component, the largest vacuoles also contain a centrally located dense structure. The contents of such recently formed G's are less dense than those of the definitive ovarian G's. Such instance of G's formation has been observed in the Golgi complexes prevalently located in ZB. G's with the same morphological characteristics are found halfway between ZA and ZB. The definitive mor- phology of G's is achieved in the cortical cytoplasm, where they are abundant and are found in numbers appropriate to the size of the oocyte. Vesicles and cisternae are seen in close contact with the membranes of G's migrated to the cortical cytoplasm. In Fig. 7 G's appear confluent with a smooth cisterna. Small vesicles can also be observed inside the G's (Fig. 8). Origin and cytochemistry of the animal dimple granules 245

Fig. 4. Oocytes at final stages of oogenesis. G's are distributed in the cortical cytoplasm over more than one layer. They show one or two electron dense core/s (CO) and a peripheral fibrous content (small arrow), which is bound by a rim of electron-dense material (double small arrows) and separated from the G membrane by an interstice of 20 nm (arrowhead). MVB = multivesicular bodies. MI = microvilli. x 27000. Inset: enlargement of the G labelled by the large arrow, x 45000.

(B) Cytochemical characterization of G's The following observations were made on the germinative area of ovarian oocyte and on the dimple of uterine eggs, where G's are found in their definitive arrangement.

(a) Detection of protein and acid phosphatase The exposure to pronase (Fig. 9 a) of ultrathin sections of the dimple results in an extraction of the central core/s of the G's. Conversely, the peripheral granule matrix is unaffected. No acid phosphatase has been found in G's in oocytes and uterine eggs (Fig. 9 b).

(b) Detection of polysaccharide complexes G's are not stained by 1 % PTA, whereas the dimple plasma membrane, in particular its outermost layer and the glycogen are stained (Fig. 9 c). With the TCH-AgP procedure, the amount of the Ag fine granular deposit changes according to the TCH exposure time used. After 24-48 h, G's core and its 246 P. ANDREUCCETTI AND C. CAMPANELLA Origin and cytochemistry of the animal dimple granules 247 peripheral contents present some deposit of silver granules (Fig. 9d). A slightly positive reaction is still detectable in the G's after 72 h exposure to TCH.

DISCUSSION This study presents evidence concerning the origin of G's, the oogenetic stages of their formation and their cytochemistry. It has also been shown that the G's are formed exclusively in the germinative area peripheral cytoplasm. This suggests that the oocyte of Discoglossus provides an example of early segregation in a specific zone, the germinative area, of structures such as G's, which persist throughout ovulation and deposition until they are called into action at activation (Campanella, 1975; Denis-Donini & Campanella, 1977). G's form at the end of oogenesis from Golgi complexes, mostly from those located in the germinative area deeper cytoplasmic region, i.e. ZB. G's appear to derive from the gradual enlargement of vesicles pinched off from the dilated terminal end of Golgi saccules, with a sequence closely resembling the granule formation in eucariotic cells (Jamieson & Palade, 1977). The recently formed G does not appear to have achieved its final composition. This is accomplished only after the G's have migrated to the cortical cytoplasm, probably through the further fusion of cisternae and vesicles with the G. The described origin of G's is in agreement with data reported for oocytes of other species, where cortical granules originate from Golgi complexes, located in the cortical cytoplasm (Balinsky & Devis, 1963; Taddei & Campanella, 1965; Ward & Ward, 1968), as well as in a deeper cytoplasmic region (Balinsky & Devis, 1963). In particular, Ward & Ward (1968) have shown that cortical granules form from enlarged Golgi saccules and that vesicles located in the cortical cytoplasm participate in their formation. More recently Selman & Anderson (1975) have indicated that in hamster oocyte vesicles of the endoplasmic reticulum play a part in cortical granule formation. In the full grown oocyte G's are found in the cortical cytoplasm, where their location concerns one or two layers similarly to cortical granules of other species (Balinsky & Devis, 1963; Ward & Ward, 1968). In the uterine eggs they

Fig. 5. A cluster of vesicles filled with some electron-dense material is between two Golgi complexes (GC). The saccules extremities (arrowheads) are dilated and close to vesicles (small arrow) of the same electron-density. In the middle (double small arrows) a multiple vesicle confluence to a secretory vacuole (SV) is indicated. The forming face of the Golgi complexes on the right is characterized by patches of electron-dense material where vesicles are embedded (large arrows), x 45000. Fig. 6. Secretory vacuoles (SV) ranging from 0-2 to 0-7 /m\ are observed next to a Golgi complex (GC). Some of them (G) have an electron-dense core and a fibrous periphery, similarly to the typical G's structure. The large arrows indicate the electron-dense patches of the forming face, x 41000. 248 P. ANDREUCCETTI AND C. CAMPANELLA

Fig. 7. In the germinative area cortical cytoplasm many vesicles surround the G's. Aflattened vesicle is confluent with a G(G) membrane (arrow). MI = microvilli; P = pigment; MVB = multivesicular body, x 30000. Fig. 8. A small vacuole (arrow) is present in the G(G). MI = microvilli. x 30000.

Fig. 9. (a) Pronase-treated ultrathin section of the uterine egg dimple. Only the G's core is completely digested by the enzyme: compare with the G's core in the control section unexposed to pronase (inset). Y = vesicle; FB = fibre bundle; MI = microvilli; GL = antennular glycocalyx. x 29000. (b) Uterine eggfixed for acid phosphatase detection, stained with uranyl acetate and lead citrate. G's appear slightly more electron dense than in the control (inset) where the incubation in the Gomori medium has been done without /?-glycerophosphate. However, any positive reaction is detectable in G's in the section fixedfo r acid phosphatase detection. P = pigment, x 29000. (c) Uterine eggs. The ultrathin sections have been stained with PTA. Glyc- ogen (GLY) and the outermost plasma membrane layer (large arrow) are stained. G's are not stained. V = vesicles. In the inset the control sections are not exposed to PTA. The glycogen is unstained, x 54000. (d) Periodic acid - TCH-AgP-treated uterine eggs. TCH 48 h. A finegranula r deposit is detectable on the glycogen (GLY) and on G's (G) whereas in G's of the control section - not exposed to periodic acid -is absent (inset). MI = microvilli; FB = fibre bundle; V = vesicles, x 36000. Origin and cytochemistry of the animal dimple granules 249 250 P. ANDREUCCETTI AND C. CAMPANELLA are concentrated in chains distributed over a deeper cortical area (Campanella, 1975). It can bz postulated that the final distribution of G's is most probably a consequence of the organization of the dimple cytoplasm, where filament bundles traverse the peripheral cytoplasm and cause G's to distribute along the channels created by the bundles themselves. The cytochemical results suggest that G core is mainly composed of proteins. In this regard G core shows, in favourable sections, a crystalline arrangement (Campanella, 1975). The protein nature of G core is in agreement with the demonstrated presence of proteins, and, in particular, of proteases in cortical granules of Echinoderms and Mammals (Epel, 1975). Polysaccharide complexes appear to be a minor component of G's where they are probably present as mucopolysaccharides or glycoproteins for the G content is positive to the Thiery's method at the longest exposure time to TCH. By contrast, in Anurans cortical granules a high presence of polysaccharide complexes has been demonstrated through the same cytochemical method (Favard & Favard Sereno, 1969). It has also been shown in eggs from a variety of species, including Amphibian ones, that cortical granules contain sulphated mucopolysaccharides (Schuel, Kelly, Berger & Wilson, 1974). Cortical granules are probably comparable to lysosomes (Brachet, 1960) in their origin from Golgi complexes, enzymatic content and structure-linked latency and activation (see Schuel et al. 1972). In G's of Discoglossus the lysosomal marker enzyme acid phosphatase is not detected, similarly to data reported for Echinoderms and Mammals cortical granules (see Anderson, 1974). This result is not in favour of a lysosomal nature for G's. As already mentioned, a successful sperm penetration occurs only at the animal dimple, which is also the only egg site able to respond to the activating stimulus with G exocytosis (Campanella, 1975). Anurans cortical granules are found in the whole egg cortex, and spermatozoa can successfully penetrate, no matter where, the animal hemisphere (Elinson, 1975). Thus the relationship between cortical granule presence and fertilization is stressed in Discoglossus by the fact that G's form and are maintained only in the zone destined to become the site of sperm-egg interaction. In conclusion, we propose that G's are cortical granules as they seem to correspond to cortical granules of other Anurans on the basis of their location, com- mon origin from Golgi complexes and release of their contents at fertilization. It should be taken into account, however, that they probably have a different macromolecular content as compared to the Anurans cortical granules and originate from Golgi complexes of oocytes in advanced stages of oogenesis whereas cortical granules derive from Golgi complexes, when the oocytes are in the early stage of oogenesis (Balinsky & Devis, 1963; Ward & Ward, 1968). Origin and cytochemistry of the animal dimple granules 251 We are grateful to Prof. G. Ghiara and Dr A. Tartakoff for suggestions and revision of the manuscript and to Dr V. Graziano for her assistance. We also wish to acknowledge the expert assistance rendered by the 'Centro di Studi di Microscopia Elettronica' of the Uni- versity of Naples. This work was supported by a C.N.R. grant of the 'Biology of Reproduc- tion Project'.

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