Further Notes on the Gametogenesis and Fertilization of Sponges. Prof. J

Further Notes on the Gametogenesis and Fertilization of Sponges. Prof. J. Bronte Gatenby,M.A.,Ph.D. (Dubl.), D.Phil. (Oxon.), D.Sc. (Lond.), Trinity College, Dublin. With Plates 19-21. INTRODUCTION.1 MY previous work on the sponge Grantia was incomplete in several respects, because even after some years' work all the required stages had not been found. I could not satisfy myself as to the nature of the granules in the choanocytes, other than the Golgi element, which was correctly identified. It was not possible to make a report on the early changes in the choanocyte and spermatozoon after the entry of the latter into the former. The oogenesis was not properly studied because in none of my successful Golgi preparations did eggs occur. The early and late stages of spermatogenesis were not discovered. The discovery of the behaviour of the coarse granules found in the choanocytes of Grantia compressa, and figured by Minchin and Dendy, has enabled me to identify such granules as mitochondria. In my previous paper I called them ' mitochondria and yolk ' in an uncertain manner : at that time I could not feel satisfied that such irregular large granules could be mitochondria alone, because mitochondria of other inverte- brates are rarely if ever of such an appearance. The difficulty was that one could not imagine such coarse irregular granules 1 The various cell inclusions described in this present paper, and in the various parts of the ' Cytoplasmic Inclusions of the Germ-cells ', can be seen intra vitam and stained intra vitam. They are not produced by the technique used. NO. 282 N 174 J. BRONTE OATENBY taking part in the cell divisions associated with rapid growth, whereby the few coarse granules might ultimately become segregated in a few cells, and be absent in others. On the other hand, no fine equal-sized granules had previously been noted in G r a n t i a by any observer. Haeckel, Dendy, and the present author had stated their belief in the metamorphosis of collar- cells into egg-cells. If such took place it was indicated that spermatozoa might be formed in that way too. The occurrence of the coarse granules in collar-cells did not make this likely, because at the spermatid stage some cells would contain very coarse granules, others none at all, or at best very few. In no animals hitherto described do spermatids occur with widely differing quantities of mitochondrial substance, for it is evident that if such occurred the resulting spermatozoa would be likely to have middle-pieces of vastly differing sizes. The similarity of all spermatozoa of a given animal, in respect of the cytoplasmic apparatus, is a well-known fact. It was, as I have mentioned, the discovery of a peculiar fragmentation process of the choanocyte granules which solved these difficulties. PREVIOUS WORK. The only two papers devoted entirely to the gametogenesis of Grantia are those of the late Professor A. Dendy and the present writer. Dendy (1) showed that during oogenesis young oocytes engulf entire cells, which are brought to them by special carrier cells. He also described the remarkable appearance of the young eggs such as is shown in fig. 13, PI. 21, of the present paper ; the nucleolus becomes squeezed out to form lumps, which are extruded into the cytoplasm, and form what Dendy considered to be chromidia. In his fig. 52, PI. 24, he gives a drawing of what we now know to be fertilization. Dendy tacitly accepted the interpretation of Jorgensen (6), that this stage is the feeding of the oocyte by means of nurse-cells. Jorgensen's descriptions of this phenomenon had been accepted by the German cytologists, but my paper in 1919 showed clearly that this stage was the last period of entry of the swollen sperrna- GAMETOGBNBSIS OF SPONGES 175 tozoon into the oocyte. Jorgensen and Dendy missed an interesting discovery by under-differentiating their slides. Both Jorgensen and Dendy described a process whereby a part of one of the pronuclei occasionally became separated and lay in the cytoplasm as an accessory body or ' karyomere '. In my previous paper, read in December 1919 before the Linnean Society (2), I showed that the spermatozoon of the collar-cell does not penetrate directly into the egg, but passes into the collar-cell, in which it swells up considerably. In Grantia compressa it was shown that the collar-cell becomes considerably modified by the presence of the spermatozoon. In my paper at that time I made some attempt to describe the cytoplasmic inclusions. The Golgi body was correctly identified, but I was considerably puzzled by the other granules in the cell, and could not give a satisfactory account of their nature and behaviour. The embryology of the amphi- blastula of Grantia was described in detail. The description by Haeckel (4) and Dendy of the metamorphosis of collar-cells into eggs was upheld. At the meeting of the Linnean Society on that date both Professor Dendy and Dr. Bidder accepted my interpretation of the stages in fertilization and spermatogenesis described by me. It was admitted that Professor Dendy's and Dr. PolejaefFs sperm stages were either plant parasites or inquilines. TKCHNIQUE. In my previous paper I depended on the fixatives of Champy- Kull, Hermann, or my own modification of Hemming, and Kopsch's original method. I have since tried Nassonow's and Ludford's methods without much success. This work is based on the Champy-Kull fluid and Plemming without acetic (F.W.A.), followed by Heidenhain's haematoxylin. I am trying Lud- ford's methods again, for by them a more delicate control of osmium dioxide precipitation is obtainable. I have found that even after prolonged heating in osmic solution and hot water it is very difficult to get the osmium dioxide to precipitate. N2 176 J. BKONTE GATENBY CELL GRANULES IN CHOANOCYTES AND OOCYTES. It has already been mentioned that my recently prepared slides showed clearly that the polymorphic granules already figured in Grantia compressa were mitochondrial in nature. This conclusion is based on the following facts. In two areas of the sponge I have noted that much activity of the inclusions occurs, namely on the outside where new choanocyte chambers are in process of formation, and in rarer cases towards the centre of the sponge substance. In fig. 11, PL 20, is drawn, at a fairly low magnification, a part of a flagellated chamber. Normal choanocytes are at NA, while at AT are collar-cells containing more granules than is usual, an intermediate zone being marked IA. Closer examination of the choanocytes at AT shows that many contain peculiar spheres made up of from twenty to fifty minute granules. In fig. 25, PL 21, one such cell is drawn, the sphere (SPH) being a remarkable object. Now in fig. 19, PL 21, another type of flagellated cavity is shown. None of the flagellated cells has a collar, and on the basement side in every case is found a single sphere of minute granules, such as is drawn with a higher power in fig. 22, PL 21. In rare cases in this area other granules are found in the cell. One can soon find areas of collared cells which show clearly how these remarkable spheres arise. In fig. 20, PL 21, is a normal choanocyte containing a few polymorphic granules ; in Grantia practically all collar-cells will be found to be of this type. In fig. 26, PL 21, is another collarless cell from a flagellated cavity. It contains four granules, a, b, c, and another. Granules a and b are breaking up, the latter forming a number of minute granules : in fig. 24, PL 21, is another collarless choanocyte showing the same phenomenon. In fig. 21, a, b, and c, PL 21, are three stages in the fragmentation of the large granules of the collar-cells. Such stages are very clear in my material of Grantia, and I can hardly be mistaken about it. It should be pointed out that, while none of the cells in the flagellated cavity in fig. 19, PL 21, had a collar, the changes GAMETOGENESIS OF SPONGES 177 in the granules which bring about the formation of the spheres in figs. 22 and 25, PI. 21, often take place in cells with collars, as in fig. 25, PI. 21. The appearance of the granules does not therefore herald the disappearance of the collar, except in the special cases to be described below. All sponge eggs I have studied contained a fair number of chromophile granules. Sponge eggs, however, never contain so many granules as most other eggs. The chromophile granules of Grantia are often remarkable crenulated objects, as shown in fig. 8, EG, PI. 19, and fig. 12, PI. 20. In Sycon ciliatum such granules are shown in the oocyte in fig. 17, PI. 21. Dendy and others have figured these granules, the first worker calling them ' chromidia '. The origin of these remarkable- ' nucleolar emissions ' of Dendy is a matter of importance, because there is little room for doubt that these granules correspond to the mitochondria of collar-cells. Dendy quite accurately described the peculiar condition of the oogonia entering the oocyte stage, so far as concerns these granules, and since both Dendy and I considered that these bodies were nuclear in origin, it seemed interesting to re-examine the question whether a nuclear origin of mitochondria had at last been proved. Some new Sycon material has been helpful, in an unexpected manner, for I could not go any further in my investigations with the new sections of Grantia compressa.

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