Original article

Comparison between and related ovarian structures in a reptile, Pseudemys scripta elegans (turtle) and in a bird Coturnix coturnix japonica (quail)

M Callebaut L Van Nassauw, F Harrisson

RUCA, UA, Laboratory of Human Anatomy and Embryology, 171 Groenenborgerlaan, B-2020 Antwerp, Belgium

(Received 24 October 1996; accepted 27 January 1997)

Summary ― The aspect of the oogonia during their premitotic DNA synthesis and of the premeio- cytes during their premeiotic DNA synthesis was studied in turtles by autoradiography, after injec- tion of 3H-thymidine. As in the adult laying quail, the intrafollicular oocytes of the adult turtle go through three successive stages: prelampbrush, lampbrush and postlampbrush. During the prelamp- brush and lampbrush stage two kinds of nucleoli exist: peripheral and central. In contrast to avian , during its final rapid growth, no polyhedric protein yolk units were found in turtle yolk. As in the yel- low yolk of quail, highly osmiophilic alcohol insoluble satellite yolk ( oil) accumulates between the protein globular yolk of Pseudemys. Turtle yolk globules increase in volume by fusion. The pen- etration of peripherally assembled yolk in the turtle germinal disc is analogous to what we have described in the quail. Also in postlampbrush germinal discs subcortical ooplasmic organelles are pre- sent. Below the turtle germinal disc no structure comparable to the avian nucleus of Pander could be observed. No pyriform cells (as in squamate reptiles) and no pyriform-like cells (as in birds: Calle- baut, 1991b) were found in the chelonian ovarian granulosa layer. We could not demonstrate func- tional lacunoperitoneal communications via openings in the hilus ovarii of the turtle as is the case in birds. / turtle / oogenesis / avian oocyte

Résumé ― Comparaison entre l’ovogenèse et structures de l’ovaire du reptile Pseudemys scripta elegans (tortue) et d’un oiseau (caille japonaise). L’aspect des ovogonies pendant leur synthèse d’ADN prémitotique et des prémeiocytes pendant leur synthèse d’ADN prémeiotique a été étudié chez Pseuderrcys par autoradiographie après injection de thymidine tritiée. Chez la tortue adulte, les ovocytes intrafolliculaires passent par trois stades successifs : avant le stade en écou-

* Correspondence and reprints Tel: (32) 03 218 03 90; fax: (32) 03 218 03 98; e-mail: [email protected] villon (prelampbrush), stade en écouvillon (lampbrush), stade après écouvillon (postlampbrush). Pendant la période de croissance rapide de l’ovocyte de tortue, on ne trouve pas de masses polyh- édriques de vitellus comme chez l’oiseau. Pendant la période de croissance finale, on trouve des gouttes de lipides hautement osmiophiles, insolubles dans l’alcool entre les globules vitellins de tor- tue. Les globules vitellins de tortue grandissent par fusion. L’assemblage du vitellus qui participe à la formation du disque germinatif est analogue à ce que nous avons décrit chez la caille. Des organites ooplasmiques sous-corticaux sont également présents. Nous n’avons pas trouvé chez Pseudemys de structure analogue au noyau de Pander tel qu’il existe chez les oiseaux. Nous n’avons pas pu démon- trer l’existence de communications entre les lacunes de l’ovaire de tortue et la cavité péritonéale. ovaire / tortue / ovogenèse / ovocytes d’oiseau

INTRODUCTION was made with oogenesis and ovarian struc- tures in birds. Only a small number of studies have been performed on oogenesis sensu latu and related ovarian structures in Chelonia: Mun- MATERIALS AND METHODS son (1904), Loyez (1906), Thing (1918), Altland Bhattacharya (1925), (1951), Guraya Female red-eared turtles (Pseudemys scripta ele- and Rahil and Narbaitz In a (1959) (1973). gans) of different ages, reared in our laboratory, previous study (Callebaut and Van Nas- were used in this study. The turtles were kept in sauw, 1987) some typical aspects of the tap water at 25-28 °C in 50 L containers under ovary of the turtle Pseudemys scripta ele- continuous infrared illumination. A stone emerg- from the water in the centre of the container gans were described. We have demonstrated ing the turtles to take a sunbath. The the existence of a well smooth permitted baby developed turtles were fed with small earth worms. After muscle-like layer (desmin immunoreactive) a few months they received quail embryos. Tur- in the ovarian tunica albuginea, giving a tles older than 1 year received newly hatched particular aspect to the follicular stigma quails. This procedure permitted us to study ooge- region. We have found that all the turtle nesis without disturbing the turtles. Adult and female turtles could be oocytes with a diameter of 200 pm or more prepuberal distinguished from the males by the presence of a shorter tail are surrounded by voluminous lacunae and and the absence of long claws on their forelegs. are in a with a enclosed follicle superficial Seven baby turtles (weight: 16-20 g) received at the surface of the crater, bulging ovary. an im injection of 100 pci methyl 3H-thymidine Also in the turtle ovary, smooth muscle-like (Amersham; 82 Ci/mmol) in water, five 1-year- cells were found in a suspensory apparatus old turtles (weight: 130-150 received an im formed by chordae, the tunica albuginea, injection of 500 pci methyl H-thymidine (82 and the theca extema of the ovarian folli- Ci/mmol). Six young adult female turtles (weight: 850-1 000 received im of 5 mCi cles Nassauw et excellent g) injections (Van al, 1991). An L-[3,5-3H] Tyrosine (Amersham, 51 Ci/mmol) review of and fol- comparative oogenesis during February or the beginning of March (just liculogenesis in birds and reptiles has been before or at the moment of egg laying under our published by Guraya (1989). In the present experimental conditions). The turtles were killed work, we have studied some aspects of oog- 1 h to several days after the injection(s). Baby turtles or turtles still with a soft cara- enesis sensu latu and related ovarian struc- 1-year-old pace were decapitated by a transverse section tures in the turtle Pseudemys scripta ele- through the middle of their carapace. Older tur- This was at different gans. performed ages, tles with a hard carapace were, first anesthesized using histochemical and autoradiographic by an im injection of 2-6 mL Nembutal methods. Whenever possible a comparison (60 mg/mL) solution in saline, before decapita- tion. After removal of the ventral part of their The radioactively labelled and some control carapace and the opening of the abdomen, the sections (PAS or Feulgen stained or not) were were fixed in situ (for baby turtles) or dipped in nuclear emulsion L4 (Ilford, UK). After removed by sectioning through the hilus ovarii 5 weeks of exposure in the dark, the autoradio- and then fixed. The volume of the ovaries varies graphs were developed according to Caro and enormously and depends on the age and physio- Van Tubergen (1962). The unstained autoradio- logical or hormonal state of the turtle. In the adult graphs from calcium formalin fixed tissues were turtle both ovaries are functional in contrast to coloured with Unna. The tissue sections obtained birds, and during the egg laying period they con- after other fixations were stained with iron hema- tain follicles of all sizes with a maximum dia- toxylin and eosin. Since Callebaut ( 1979a,b; meter of approximately 20 mm. Several large 1988a) demonstrated the existence in birds of follicles of the same diameter are seen. In contrast lacunoperitoneal communications, functional via to birds both ovaries of turtles (and reptiles in the hilus ovarii we decided to investigate if this general) contain permanent groups of extrafol- was also the case in Pseudemys scripta elegans. licular germ cells (oogonia and oocytes) which To do this, finely divided yolk of five unincu- are called germinal centres or beds. Using char- bated quail were suspended in 1.5 mL coal marks, the germinal disc of the largest oo- Ringer solution containing 1 % trypan blue (Van cytes was labelled. The oocytes of approximately Nassauw and Callebaut, 1991). From this sus- the same diameter were fixed in different fixati- pension 0.3-0.5 mL were injected intraperi- ves for comparison. Fixation was performed toneally ventrally from the hindleg via the open- overnight at room temperature in Heidenhain’s ing in the carapace in four baby turtles, or 10 Susa without sublimate (Romeis, 1948), in cal- mL were injected in the same way into three cium formalin for 1 night at room temperature adult turtles. Five to 16 hours later the turtles or in a mixture of 2% paraformaldehyde and were killed and their ovaries fixed in Susa 0.1 % glutaraldehyde in 1.0 mM phosphate (Romeis, 1948). After fixation for 2 days at room buffered saline for 4 h at 4 °C. Other oocytes or temperature in this fixative, the ovaries were part of the ovaries were fixed in Bouin for I night placed directly in 96% alcohol for 3 days. After at room temperature followed by a postfixation in further dehydration in absolute alcohol and clea- 70% alcohol (Callebaut et al, 1991) or rinsed di- ring in xylene they were embedded in paraffin. rectly in tap water to study the presence of lipids After sectioning, the sections were deparaffinized in the oocytes during vitellogenesis. Some of the and counterstained with Kernechtrot. oocytes, fixed in Bouin and postfixed in 70% alcohol or not, were rinsed in distilled water and placed in a 1 % solution of osmium tetroxide in water for I h, followed by rinsing in tap water. RESULTS The germinal disc of the largest oocytes was excised and of were in pieces ovary dehydrated The general aspect of the baby or 1-year- the alcohol series, cleared in xylene and embed- old turtle ovary is shown in a transverse sec- ded in paraffin. After sectioning at a thickness tion at the sur- of 8-10 0 pm, most of the slides were prepared (fig 1): larger oocytes bulge for autoradiography. face. At least on each side of the mesovarium a germinal bed is visible. How- Lipids were studied on the sections without ever, by the growth of intra follicular and without Indeed dur- deparaffination staining. oocytes in the neighbourhood, these germi- ing a study of in avian follicles we observed lipids nal beds seem to become divided. A that a second passage through xylene (for depa- long, narrow mesovarium fixed to a narrow linear raffination) removes all lipids (Callebaut, 1988b). Some of the sections were Feulgen stai- hilus ovarii is seen. Openings are not visible ned, according to Demalsy and Callebaut (1967). in the mesovarium, or in the hilus ovarii. For the study of the labelling of cytogical de- After an intraperitoneal injection of trypan were take of some of the secti- tails, photographs blue-labelled we found no before and after the yolk, yolk gran- ons, autoradiographic proces- ules in the lacunae of the and ovary, indicating sing (Callebaut Demalsy, 1967). Comparable . that there is no functional communication sections of similar oocytes of non-injected females were used as controls both before and between the peritoneal cavity and the ovar- after the autoradiographic procedure. ian lacunae. After im injection of 3H-thymidine labelled germ cells of the latter type increases in the because in the baby or 1-year-old turtle germinal beds, they differentiate after divisions from pre-exist- labelled into Shortly after injection (l h), groups of oogo- ing oogonia primary oocytes nia localized in germinal centres directly (fig 4). The intensity of the labelling per nu- below the ovarian surface epithelium (or cleus, however, decreases but finally remains since these divisions sometimes on the surface) but superficially constant, end with a final division. The to the tunica albuginea, are seen to be premeiotic aspect of the nucleoli can labelled mainly on the peripheral part of most clearly be seen in sections their nuclei (containing a fine reticular Feul- through germinal beds of 1-year- gen positive network with positive chro- old turtles after staining of the sections with matin granules) surrounding a non-labelled iron hematoxylin and eosin before autoradio- central nucleolar area (compare fig 2A, graphy (fig 5). We observed a typical before autoradiography and fig 2B, after chromosome-free clearer area of nucleo- autoradiography). This labelling of oogo- plasm surrounding each of the nucleoli. With nia is due to premitotic DNA synthesis dur- this staining procedure two kinds of nucle- ing their multiplication period. But also we oli can be discerned: (1) intensely red presume that premeiotic DNA synthesis (eosinophilic) stained nucleoli, usually local- takes place in some larger germ cells that ized centrally in a chromosome-poor clear are already partially surrounded and separa- area, (2) dark stained nucleoli. This aspect of ted from each other by prefollicular cells the nucleoli is even more pronounced when (fig 3). In these germ cells, which form a the oocyte grows: the central red stained transition stage from oogonia to primary (eosinophilic) nucleoli become surrounded oocytes, relatively voluminous central nucle- by a large fine granular nucleoplasm, free oli are seen. During the days following an from chromosomes, whilst the vacuolized injection of 3H-thymidine, the number of dark stained nucleoli (only surrounded by a clear halo) are localized at the inner side of the germinal vesicle penetrates the deeper the germinal vesicle membrane (fig 6). part of the cortex where the beginning of the assemblage of small yolk spheres takes place. At the beginning of the postlamp- of the Cytology oocytes brush stage, the nucleoli are no longer loca- in the adult turtle ovary lized at the inner side of the germinal vesi- cle wall but deeper at some distance on a As in the of adult we oocyte laying quail sphere surrounding the shortened chromo- can distinguish three successive develop- somes in the central area (fig 11 When the mental of intrafollicular oocytes in stages vesicle the cortex, a adult turtles before . germinal penetrates ger- minal disc forms. In this germinal disc we The with chromo- (1) prelampbrush stage see the development of subcortical oo- somes in diplotene without clearly defined plasmic organelles (fig 12). These organelles lateral and with individual lampbrush loops are most clearly visible after Bouin fixation, nucleoli surrounded by a clearer chromo- osmium tetroxide treatment and Unna stain- some-free halo In the a volu- (fig 7). ooplasm ing and are pressed between the yolk gra- minous Balbiani close paranuclear complex nules, as is also the case in the quail. In the to an eccentric germinal vesicle could be 10-mm-diameter oocyte, the germinal vesi- seen (figs 7-9). At some distance from the cle has penetrated through the cortex (fig surface a can be obser- lipid granular layer 13). Only a narrow cap of small yolk gran- whilst at the surface of the Balbiani ved, ules separates it from the vitelline mem- a also seen. complex lipid-containing layer is brane. The short chromosomes are hardly Between both the PAS lipid layers intensely visible among the voluminous vacuolized is visible staining ooplasm (fig 9). nucleoli, aggregated in the centre of the ger- (2) The lampbrush stage with chro- minal vesicle forming an oval caryosphere. mosomes presenting the lampbrush chro- At its maximum of development, the oocyte mosome configuration with lateral loops as (at the end of the postlampbrush stage, just already described by Loyez (1906) in Cis- before maturation) reaches a diameter of tudo europaea and with numerous volumi- 19-20 mm and the germinal vesicle is found nous pyroninophilic nucleoli against the flattened at the surface of the ooplasm (fig inner side of the germinal vesicle wall 14). However, the yolk cap above the ger- (fig 10), often bulging at its surface. The minal vesicle is still present. The smaller germinal vesicle has a central or paracen- nucleoli are still visible as a flat group in tral localization and increases strongly in the centre of the germinal vesicle. The This when volume. stage begins the oocyte prominent presence of nucleoli seems thus has a diameter of 600-700 pm. During the to be a characteristic feature of the chelo- lampbrush stage, two kinds of nucleoli can nian oocyte during its whole intrafollicular be obvious after distinguished (particularly period. This is not the case during the cor- Bouin fixation, alcohol postfixation, osmium responding period in avian oocytes. In the tetroxide treatment and Unna staining) intrafollicular Pseudemys oocytes, after the (fig 10): (a) large peripheral py- Feulgen staining, the chromosomes are not visible at the roninophilic nucleoli, already or very faintly visible. Particularly during of the the beginning lampbrush stage; (b) the postlampbrush stage no spherical con- much smaller stained nucleoli close to grey tracted chromosomes are seen, as is the case or on the chromosomes, localized in the in the quail (Callebaut, 1973a). Although at large central part of the germinal vesicle. the end of their final growth period (end of (3) The postlampbrush stage starts when the postlampbrush stage), both the quail and the oocyte has a diameter of 3-4 mm and turtle oocyte reach the same maximal diam-

eter (approximately 20 mm), the prelamp- pressed against the outer side of the granu- brush stage in the turtle develops to a greater losa basement membrane. A paranuclear diameter (600 pm), than in the quail (only lipid drop could be seen in the granulosa 150 Lun diameter: Callebaut, 1975). Also cells and sometimes between the granulosa the lampbrush stage in the turtle becomes cells. After Bouin fixation and rinsing in more voluminous mm contra (3 diameter) water the satellite yolk is no longer visible. mm in the 1.5 quail. During the final growth It also from the sections after de- in the of disappears period oocyte Pseudemys scripta paraffination by xylene (Callebaut, 1988b). elegans alcohol insoluble satellite yolk or This yellow stained satellite yolk seems to egg oil (as described in birds: Callebaut et al, contribute to the colour of the 1991) localized between the protein yolk yellow larger globules (fig 15), could be demonstrated by oocytes in Pseudemys. After ip injection of fixation in Bouin followed by postfixation in trypan blue-labelled yolk in adult turtles we alcohol 70% and osmium tetroxide treat- found no yolk granules in the lacunae of the in ment. As in birds, lipid drops were also ovary, which indicates that also the adult found in the theca intema. An accumulation female no functional communications exist of large clumps of intensely staining osmiop- between the peritoneal cavity and the ovar- hilic material was also locally observed, ian lacunae. Adult turtle ovary, oocytes (approximately 4 mm in diameter), after an im injection of 3H-tyrosine, a broad labelled cortex containing small 38 h before fixation labelled yolk granules is seen (fig 20). Below this cortex, larger unlabelled yolk The labelling on the autoradiographs was spheres are seen. Small labelled granules most clearly seen after Bouin fixation and adhere to the surface of this unlabelled yolk Feulgen staining before dipping. The spheres, and seem to fuse with them after radioactive labelling was, however, no penetration through the cortex. In the neigh- longer visible in Feulgen stained sections bourhood of the germinal vesicle, which has after tissue fixation in Susa without subli- penetrated the subcortical yolk layer, the mate, calcium formalin or paraformalde- first Anlage of the germinal disc forms hyde and glutaraldehyde. After calcium for- (fig 21). malin fixation, staining with Unna after the from and below the autoradiographic processing also gives a Laterally germinal vesicle, labelled are visible good contrast with the autoradiographic yolk granules labelling. (comparable with the first Anlage of the so- called polar granules in birds: Bartelmez, of the nuclei of the extrafol- Labelling 1912, Callebaut, 1974) suggesting a sliding licular cells as clearer areas germ (visible movement. In 8-10-mm-diameter postlamp- in the is seen The germinal beds) (fig 16). brush oocytes (exterior to the germinal disc of the vesicle of the labelling germinal region) we see that the intensely labelled smallest intrafollicular oocytes (prelamp- large yolk globules are localized below the brush is intense and visible even stage) very less or non-labelled smaller yolk globules at low magnification (fig 17). Also a less (fig 22). This indicates that the 3H-tyrosine dense is seen on the whole labelling ooplasm pool was already exhausted before fixation. at higher magnification (fig 16). In some- In the germinal disc region of 8-10-mm- what larger oocytes (lampbrush stage, eg, diameter postlampbrush oocytes, the pene- 700 pm diameter) the central ooplasmic tration of the peripherally assembled labelled mass a whilst the presents labelling periph- yolk granules seems to be impaired by the eral cortex is not or labelled poorly (fig 18): presence of weakly stained subcortical this indicates a central, perinuclear metabolic ooplasmic organelles (fig 23). activity (protein synthesis). In still larger oocytes (lampbrush chromosome stage), Sometimes the labelled penetrating yolk small yolk globules begin to accumulate granules form a cap over these organelles, as below the cortex, and the labelling in the was also seen in the quail germinal disc latter increases. From this moment on, there (Callebaut, 1974). In the germinal disc are clearly two kinds of labelling from dif- region of postlampbrush oocytes of approx- ferent origins: (1) the central extravitelline imately 15 mm in diameter, we see that the labelling (most pronounced around the ger- labelled yolk layer just reaches the angles minal vesicle), and (2) the peripheral of the germinal vesicle (fig 24) without slid- labelling localized on the yolk globules ing laterally from it. Above the germinal forming a subcortical layer. Some larger vesicle a narrow yolk cap is seen composed more centrally localized yolk globules are of two parts: the deepest part, close to the not labelled and are obviously already germinal vesicle is unlabelled (and thus formed before the injection. At this moment formed first) whilst the superficial part is the germinal vesicle strongly increases in labelled and formed after the injection of volume and the nucleoplasm and the large 3H-tyrosine. The penetration of yolk into vacuolized peripheral nucleoli are intensely this region is thus strongly reduced. In the labelled (fig 19). In early postlampbrush largest postlampbrush oocytes, exterior to

the germinal disc, we can see that, at the as has been described during the final rapid boundary of labelled and unlabelled yolk, growth period in the oocytes of birds (Perry large yolk globules often have a partially and Gilbert, 1985; Callebaut et al, 1991). ie, that polarized labelling pattern, they pre- Therefore, we prefer not to speak of yolk sent a radioactively labelled cap directed platelets because the protein chelonian yolk towards the surface of the oocyte (fig 25). is present in a globular form. This seems Sometimes also contain large yolk spheres also to be the case in oocytes of other rep- one or more round central unlabelled parts, tiles, eg, in the lizard Sceloporus torquatus whilst the surrounding periphery of the yolk torquatus (Uribe et al, 1995). In the largest sphere is strongly labelled (fig 26). Even in postlampbrush oocytes the yolk cap above the largest oocytes of Pseudemys scripta the germinal vesicle no longer contains elegans, only round yolk units (yolk spheres) labelled yolk, which indicates that here the are assembled and no polyhedric yolk units, penetration of yolk precursors is negligible or absent (fig 27). The narrow labelled yolk 1991 a). In contrast to the intense labelling layer in the germinal disc ends at the angles found in the germinal vesicle of prelamp- of the very flattened germinal vesicle. The brush and lampbrush turtle oocytes, during granulosa cells above the germinal vesicle the postlampbrush stage, no or very slight are very flat whilst more laterally in the labelling is seen. During the latter period immediate neighbourhood, they form a two the germinal vesicle no longer increases in nuclei thick layer (figs 27 and 28). This is volume and the metabolic activity of the exceptional since it is usually assumed that chromosomes and nucleoli decreases con- the turtle granulosa layer is only one cell siderably. thick. The labelling in the granulosa layer is extranuclear, both above the germinal disc region (fig 28) and exterior to this region Adult turtle ovary (fig 25). This suggests that the transport of after two im injections of3H-tyrosine, protein yolk precursors mainly occurs via 4 and 8 days before fixation the intercellular spaces as is the case in the quail (D’Herde and Vakaet, 1992) and Labelling in the smaller turtle oocytes is eventually through the granulosa cytoplasm similar to the autoradiographic observations as also observed in the quail (Callebaut, after a single injection. In larger oocytes with the beginnings of peripheral protein lampbrush oocytes is the labelled yolk cap yolk assemblage, two labelled layers can be over the unlabelled yolk, localized just recognized below the relatively broad cortex superficially to the central part of the ger- (fig 29). In still larger oocytes (beginning minal vesicle (fig 32). This again indicates postlampbrush stage), the two labelled yolk that the penetration of yolk above the ger- layers become localized in a more superficial minal vesicle is very slow (more than 8 position (fig 30). Finally in the largest post- days) and there is only one labelled layer lampbrush oocytes (where the cortex is visible above the germinal vesicle. The pro- absent) the last-formed labelled yolk layer is gressive sliding of the yolk layers below the localized on the surface of the ooplasm rims of the germinal vesicle is also very (fig 31). Most obvious in the largest post- slow (8 days or more). This sliding of the deep yolk layers below the germinal vesicle the turtle. A large column extends from the is the origin of the polar granules (fig 32) centre of the oocyte to below the centre of which are analogous to the polar granules the germinal vesicle (fig 33). in birds (Bartelmez, 1912; Callebaut, 1974). The formation and the structure of the ger- The alternating morphologically different minal disc of Pseudemys scripta elegans yolk layers end perpendicularly to and lat- seems to be approximately analogous to erally from this column. After fixation with what has been described in the quail (Calle- calcium formalin and Unna staining of the baut, 1974, 1975). The sliding is, however, sections after the autoradiographic process- slower than in the since in the quail oocyte ing, we could find at least 14-15 different latter, after 8 days, the labelled layer was layers alternatively composed of the two found deep in the nucleus of Pander or in types of yolk and disposed in concentric the latebra neck. layers encircling at a large distance the cen- tre of the oocyte (figs 14 and 33). This The nucleus of Pander, first described in ressembles the banding pattern described birds by Pander (1817) as a large cushion- by Cuellar ( 1971 ) in the parthenogenetic like mass of white yolk below the germinal lizard Cnemidophorus uniparens and by vesicle, is however not clearly defined in Uribe et al ( 1996) in Ctenosaura pectinata. Comparison of the thickness and number of exist in the chelonian ovary. Our study also labelled yolk layers in the oocytes of dif- indicates that before the chelonian oocyte ferent diameter suggests that 2-3 such lay- matures, it develops through three successive ers are laid down in approximately 8 days. stages: prelampbrush, lampbrush and post- As in the of birds Consequently, we may conclude that the lampbrush. oocytes (Cal- period of relatively rapid growth in the stud- lebaut, 1973a) there exists a correlation ied chelonian oocyte lasts longer than in the between the aspect and localization of the quail oocyte, although both finally reach germinal vesicle and the ooplasm in the immediate For instance in approximately the same volume. neighbourhood. the prelampbrush stage, a voluminous paranuclear Balbiani complex is present. DISCUSSION The postlampbrush stage begins exactly at the moment when the germinal vesicle into the cortex. In the By contrast to the presence of several ger- migrates lampbrush the vesicle increases minal beds in the Pseudemys ovary, no ger- stage, germinal strongly in volume in relation to the minal beds occur in the adult avian ovary. probably lamp- brush chromosome and the Indeed in the latter all the oogonia are trans- activity appe- formed into primary oocytes at the end of arance of giant nucleoli. The labelling in embryonic and the beginning of postnatal the germinal vesicle after 3H-tyrosine injec- tion is most the life. In birds there are no stem germ cells pronounced during prelamp- for repeating the seasonal multiplication of brush and lampbrush stages, whilst the oogonia by mitosis. The premitotic and pre- labelling in the postlampbrush germinal meiotic DNA synthesis in the chelonian vesicle is minimal or absent and its volume in oogonia and premeiocytes have some remains constant. Our study indicates that homology with what we have described in the chelonian oocyte there also exist sub- and quail, respectively (Callebaut, cortical ooplasmic organelles distributed at 1967, 1973b). The labelling after 3H-thymi- the animal pole around the germinal vesi- dine administration is localized on the Feul- cle. These organelles present homology gen positive chromatin threads surroundingg (similar aspect, relationship with penetrating the central nucleoli. A difference with yolk and development at a comparable respect to the corresponding developmen- stage) with the geometrically distributed tal stage in birds is that the germinal beds subcortical cytoplasmic organelles or ticos in the turtle are much less voluminous. In in the quail oocyte (Callebaut, 1972, 1983; the turtle, oogonia or preoogonia are often D’Herde et al, 1995), and with the radially not localized below the tunica albuginea of disposed mitochondrial clusters described the ovary but still more superficially at the by Tourte et al (1984) in the Xenopus laevis surface of the ovary in direct contact with the oocyte. In the present study, at the begin- peritoneal cavity (fig 5). This very superfi- ning of peripheral yolk formation, we cial position constitutes perhaps a migra- demonstrated that after penetration through tion path of undifferentiated female germ the oocytal cortex, granules adhere to the cells on the ovarian surface. Indeed since deeper, larger and earlier formed unlabelled the studies of Waldeyer (1870) we know yolk globules. This observation visualizes that in most female reptiles (in contrast to the fusion hypothesis of Ho (1987) and birds and mammals) oogonia persist until Guraya (1989). Moreover, the polarized or after sexual maturity and give rise to oocytes partially superficial radioactive labelling by mitotic divisions (Zuckerman, 1962). A after 3H-tyrosine administration often seen follicular hierarchy with a limited number of in the large yolk globules of the large post- follicles as in the avian ovary seems not to lampbrush oocytes also provides evidence for this fusion hypothesis. In the largest porospatial distribution, that has much anal- Pseudemys postlampbrush oocyte, we found ogy with the pyriform cells described in no particular yolk structure similar to the squamate reptiles. We found no such pyri- nucleus of Pander (1817), visible in birds. form-like cells in the chelonian granulosa Even in the largest Pseudemys postlamp- layer. The peripheral arrangement of many brush oocyte, we could (in contrast to the nucleoli in the previtellogenic oocytes of avian oocytes) distinguish morphologically fish, amphibia and turtles suggests some the successive yolk layers in the deeper yolk phylogenetic relationships among them mass below the germinal disc by the use of (Guraya, 1989). By contrast the oocytes of an appropriate fixative (calcium formalin) squamates (Arronet, 1973) and of birds and Unna staining (fig 33). So it can be seen (Callebaut, 1975) have a few nucleoli that that the successive yolk layers in Pseude- are localized in the central karyoplasm. This mys form only concentric spheres around is a second similar aspect of the follicular the centre of the oocyte. These layers do not and oocytal structures in squamate reptiles circumscribe a second centre (nucleus of and birds. Moreover, the obvious and poly- Pander) localized below the germinal disc as morphic development of the granulosa layer we have demonstrated in the quail oocyte in squamates (with a.o. pyriform cells) and (Callebaut, 1974, 1975) by autoradiographic in birds (with pyriform-like cells: Callebaut, studies. In the parthenogenetic lizard, Cne- 1991 b) seems to be inversely proportional to midophorus uniparens, Cuellar (1971) the feeble nucleolar development in the ger- described that the germinal disc lies more minal vesicle of their oocytes. By contrast in or less isolated from the rest of the yolk. Chelonians the granulosa remains monomor- This does not seem to be the case in the che- phic and mainly single-layered, whilst the lonian oocyte where we observed in the pre- nucleoli are very large and numerous and sent study that there is always continuity persist throughout the oocytal development. between the smaller radioactively labelled Perhaps only the central nucleoli observed layers in the germinal disc and the broader during the prelampbrush and the lampbrush superficial layers exterior to the germinal stage in Pseudemys are homologous to the nucleoli seen in aves and in disc. Among Sauropsidia, a remarkable dif- squamate rep- tiles. ference in the structure of the ovarian gran- ulosa has long been recognized (Loyez, In the present study we demonstrate, by ip 1906). In turtles, it consists, throughout the injection of trypan blue-labelled yolk, that no follicle growth of a simple, homogenous functional communications exist between cuboidal or columnar epithelium in which no the lacunae of the chelonian ovary and the pyriform cells were described (Munson, peritoneal cavity. By contrast, in the quail it 1904; Loyez, 1906; Thing, 1918; Callebaut has been shown by different labelling pro- and Van Nassauw, 1987). In contrast, the cedures that lacuno peritoneal commu- granulosa of lizards and snakes (Squamata) nications are present (Callebaut, 1979a,b, develops into a complex layer composed of 1988a). The reason for the difference is not several cell types (Loyez, 1906; Betz, 1963; known. Between the hilus ovarii of both Varma, 1970; Uribe et al, 1995, 1996). The species there are indeed obvious differences. heterogeneity of the granulosa cell popula- In birds the hilus ovarii is very irregular in tion in squamate reptiles is unlike that form. Moreover, the avian ovary is fixed, known for any other vertebrate: most notable without a mesovarium, directly to the dorsal is the transitory presence of very large clear wall of the peritoneal cavity in the imme- pyriform cells. In the quail granulosa layer diate neighbourhood of the vena cava infe- we have described pyriform-like cells rior. The eggs in birds are laid one by one, (Callebaut, 1991b) with a structure and tem- sometimes over long periods. The rapid growth phase of the avian oocytes (end of Callebaut M ( 1974) La formation de l’oocyte d’oiseau. the is faster than in Étude autoradiographique chez la caille japonaise postlampbrush stage) pondeuse a I’aide de la leucine triti6e. Arch Biol turtle oocytes. This requires perhaps the (Bruxelles) 85, 201-233 existence of an and expansion space escape Callebaut M ( 1975) Bijdrage tot de studie van de ooge- route for the increasing pressure in the ova- nesis van de vogels. PhD thesis. RUCA, Antwerp, ry. Belgium Callebaut M (1979a) Lacunoperitoneal communica- tions in the ovary of the baby quail. IRCS Med Sci 7,384 ACKNOWLEDGMENTS Callebaut M ( 1979b) The avian ovary is an open organ; a study of the lacunar system. Anat Embryol 158, We thank S Van Rompaey and V Van der Stock 103-119 for excellent technical assistance, F De Bruyn Callebaut M (1983) Electron microscope study of ticos for artwork and V De Maere for typing the (3H-thymidine incorporating cytoplasmic manuscript. This study was supported by grant organelles) in the germinal disc of the postlamp- 3.0029.93 of the National Fund of Scientific brush oocytes of Japanese quail. IRCS Med Sci 11,I, 491-492 Research Belgium and a matching fund from the University Centre of Antwerp (RUCA). Callebaut M ( 1988a) The ovarian chordolacunar system in birds. Arch Biol (Brussels) 99, 1-IS Callebaut M (1988b) Localization of voluminous highly osmiophilic material in the largest ovarian follicles REFERENCES of laying Japanese quails. Med Sci Res 16, 1307- 1309 Altland P ( 1951 ) Observations on the structure of the Callebaut M (1991a) Electron microscopic evidence reproductive organs of the box turtle. J Morphol for the delivery of lipids by the granulosa cells to the 89,599-6211 superficial avian ooplasm. Eur Arch Biol (Brus- Arronet VN (1973) Morphological changes of nuclear sels) 102, 47-53 structures in the oogenesis of reptiles (Lacertidae, Callebaut M (1991b) Pyriform-like and holding gran- Agamidae). J Herpetol7, 163-193 ulosa cells in the avian ovarian follicle wall. Eur Arch 135-145 Bartelmez G (1912) The bilaterality of the pigeon’ss Biol (Brussels) 102, egg. A study in egg organization from the first Callebaut M, Demalsy P (1967) Improved resolution of , growth period of the oocyte to the beginning of fine caryological details by staining and examina- cleavage. J Morphol 23, 269-329 tion before and after autoradiography. Stain Tech- Bhattacharya DR ( 1925) Les inclusions cytoplasmiques nol 42, 227-229 dans 1’oogenese de certains reptiles. These. Vign6 Callebaut M, Van Nassauw L (1987) Demonstration by Ed, Paris, France monoclonal anti-desmin of a myoid tissue coat in Betz TW (1963) The ovarian histology of the diamond- the preovulatory ovarian tunica albuginea of the backed water snake, Natrix Rhombifera, during the turtle Pseudemys scripta elegans. Med Sci Res 15, reproductive cycle. J Morphol 113, 245-260 1129-1130 Callebaut M (1967) Premeiosis and premeiotic DNA Callebaut M, D’Herde K, Hermans N, Van Nassauw L Localization and of in the synthesis in the left ovary of the female chick (1991) transport lipids embryo. J Embryol Morph 18, 299-304 avian ovarian follicular layers and the structural relationship of theca and granulosa to the basement Callebaut M (1972) Cytoplasmic uniaxial!radial sym- membrane. J Morphol 209, 143-163 metry during the final growth period in the oocytes of the Japanese quail (Coturnix coturnix japonica). Caro L, Van Tubergen R (1962) High resolution autora- Experientia 28, 62-63 diography. J Cell Biol 15, 173-188 Callebaut M (1973a) Correlation between Cuellar 0 (1971) Reproduction and the mechanism of germinal meiotic restitution in the lizard vesicle and oocyte development in the adult parthenogenetic Japanese quail (Coturnix coturnix japonica). A Cnemidophorus uniparens. J Morphol 133, 139- cytochemical and autoradiographic study. J 166 Embryol Exp Morph 29, 145-157 Demalsy P, Callebaut M (1967) Plain water as a rins- Callebaut M (1973b) La synth6se de 1’ADN ing agent preferable to sulfurous acid after the Feul- pr6mito- nuclear reaction. Stain tique et pr6meiotique dans les cellules germinales gen Technol 42,133-136. de 1’embryon femelle de la caille japonaise D’Herde K, Vakaet L (1992) Study of yolk precursor (Coturnix cotumixjaponica). Experientia 29, 471- transport in the avian ovary with the use of horse 472 radish peroxidase.IntJ Dev Biol 36, 435-438 D’Herde K, Callebaut M, Roels F, De Prest B, Van Romeis W (1948) Mikroskopische Technik 15. Leib- Nassauw L (1995) Homology between mitochon- nitz, Aufl Munchen, Germany driogenesis in the avian and amphibian oocyte. Thing A (1918) The formation and structure of the Dev 305-311 Reprod Nutr 35, zona pellicida in the ovarian eggs of turtles. Am J Guraya SS (1959) Histochemical studies of lipids in Anat 23, 237-258 oocytes VII. Lipids in the oogenesis of the fresh- Tourte M, Mignotte F, Mounolou JC ( 1984) Hetero- water turtle Res Bull Lissemys punctata. Panjab geneous distribution and replication activity of Univ 10, 305-313 mitochondria in Xenopus laevis oocytes. EurJ Cell Guraya SS (1989) Ovarian Follicles in Reptiles and Biol34, 171-178 Birds. Springer Verlag, Berlin, Germany Uribe M, Mendez Omana M, GonzAlez Quintero J, Ho SM (1987) Endocrinology of vitellogenesis. In: Guillette L Jr (1995) Seasonal variation in ovarian Horntones and Reproduction in Fishes. Amphibians histology of the viviparous lizard Sceloporus and Reptiles (DO Norris and RE Jones, eds), Ple- torquatus torquatus. JMorphol226, 103-1199 num Press, New-York, USA, 145-170 Uribe M, Portales GL, Guillette L Jr (1996) Ovarian fol- Loyez M (1906) Recherches sur le d6veloppement liculogenesis in the oviparous Mexican lizard Cte- ovarien des oeufs meroblastiques a vitellus nutritif nosaura pectinata. J Morphol 230, 99-1122 abondant. Arch Anat Microscop Morph Exp 8, 69- Van Nassauw L, Callebaut M (1991) Structural and 398 immunohistochemical aspects of the postovulatory Munson J (1904) Researches on the oogenesis of the follicle in Japanese quail. Anat Rec 229, 27-30 tortoise Clemmys marorata. Am J Anat 3, 311-346 Van Nassauw L, Harrisson F, Callebaut M (1991) Pander C (1817) Historiam metamorphoseas quam Localization of smooth-muscle markers in the ovum incubatum prioribus quinque diebus subit. ovaries of some ectothermic vertebrates. Anat Rec FE Nitribitt Wirceburgi 229, 439-446 Perry M, Gilbert AB (1985) The structure of yellow Varma S (1970) Morphology of ovarian changes in yolk in the domestic fowl (Gallus domesticus). J the garden lizard Calotes versicolor. J Morphol Ultruct Res 90, 313-322 131, 195-210 W Eierstock und Ei. Rahil KS, Narbaitz R (1973) Ultrastructural studies on Waldeyer (1870) Engelmann, the relationship between follicular cells and grow- Leipzig, Germany ing oocytes in the turtle Pseudemys scripta ele- Zuckerman S (1962) The Ovary. Academic Press, New gans. JAnat 115, 175-186 York, USA