The Oogenesis of Calanus finmarchicus. By Irene F. Hilton. M.Sc, Lecturer in Zoology, University of Edinburgh. With Plates 9 and 10, and 5 Text-figures. CONTENTS. PAGE 1. INTRODUCTION ......... 193 2. MATERIAL AND METHODS ....... 194 3. LITERATURE ......... 195 4. GENERAL ACCOUNT ........ 196 5. NUCLEUS, NUCLEOLUS, AND NUCLEOLAR EXTRUSION . 200 6. THE MITOCHONDRIA ........ 207 7. YOLK-FORMATION ........ 211 8. THE GOLGI APPARATUS ....... 213 9. DISCUSSION 215 10. SUMMARY 218 11. LIST OF REFERENCES. ........ 220 12. EXPLANATION OF FIGURES ....... 220 INTRODUCTION. THE present investigation was undertaken with a view to working out the details of oogenesis in a Copepod, about which comparatively little is known from the modern cytological standpoint. Calanus finmarchicus was suggested by Mrs. E. C. Bisbee, of the Department of Zoology, University of Liverpool, as a suitable subject; it is easily obtainable in quantity and has relatively large eggs; moreover the nucleolus in its be- haviour throughout the growth of the egg presents certain very interesting features. The work was carried out partly in the Department of Biology, University College of Swansea, and partly in the Department of Zoology in the University of Edinburgh. 194 IRENE F. HILTON MATERIAL AND METHODS. The specimens of Calanus finmarchicus (Gunnerus, 1770) used in the present investigation were obtained mainly from Plymouth, but also from the Marine Laboratories at Port Erin, Isle of Man, and at Millport, Buteshire. The animals were examined at intervals of about fourteen days during the greater part of two years. Some difficulty was experienced in obtaining satisfactory fixation of the ovary. Dissecting out the ovary before fixation was not found practicable but by cutting off the abdomen at the first joint before immersing in the fixing fluid, rapid penetration of the fixative was obtained with improved fixation of the deli- cate posterior end of the ovary. Memming-without-acetic acid followed by iron haematoxylin (4 or 5 hours in iron alum and overnight in 0-5 per cent, haema- toxylin) generally gave the best results, but Bouin's fluid and corrosive sublimate were found excellent as nuclear fixatives. The Champy-Kull method gave good results in the older oocytes approaching maturation. For the study of the mitochondria the methods of Kolatschev, Nassonov ('Microtomist's Vade-Mecum', 9th edition, page 345), and Champy-Kull were used. Flemming-without-acetic acid followed by iron haematoxylin was particularly successful in older oocytes. For the study of the Golgi apparatus Da Fano's method gave the best results. The methods of Nassonov, Kolatschev.. Kopsch. and Mann-Kopsch were tried without success. The author acknowledges her indebtedness to Dr. F. A. Mocke- ridge of the University College, Swansea, where the work was begun, to Professor J. H. Ashworth, of the University of Edin- burgh, to Professor W. J. Dakin, and Mrs. R. C. Bisbee, of the University of Liverpool, and Mr. L. A. Harvey, of the University of Edinburgh, for much helpful advice, and to the Committee of the Earl of Moray Endowment of the University of Edinburgh for a grant in aid of expenses. OOGENESIS OF CALANUS 195 LITERATURE. Although the Copepodaasa group have been widely studied in the past, comparatively little is known of the details of their gametogenesis from the modern cytological standpoint. Prior to the development of modern cytological methods attention was directed almost exclusively to the nucleus and its chromo- some content. The chromosome numbers for thirty-five species of Copepods are recorded in the 'Tabulae Biologicae 4' (1927). The majority of the numbers given are for members of the genus Cyclops but some species of Gymnoplea are in- cluded. Among the Gymnoplea, with the exception of a Japanese form Diaptomus sp. Ishikawa (1891) and Dia- ptomus coeruleus (Amma, 1911) the haploid number of chromosomes present in the female is either sixteen or seventeen, sixteen being the more usual number. McClendon (1906, 1910) and Kornhauser (1915) both working upon parasitic copepods described the formation of ring-shaped double chromosomes by parasyndesis in a way which appears similar to that recorded for Calanus finmarchicus in the present investigation. Matscheck (1910) in a paper upon growth and development of copepod eggs recorded fragmentation of the nucleolus prior to the maturation divisions of the egg. This author also found formation of yolk in the half-grown oocytes and suggested a possible secretory function of the nucleolus. The more recent work of Ludforcl (1922, 1924) upon the morphology and physi- ology of the nucleolus provides strong evidence in support of this view. Gardiner (1927) working upon Limulus poly- p hem us has suggested a very specialized secretory function for the nucleolus in the transport of phosphorus to the cytosome. The study of Calanus finmarchicus affords strong evidence for suggesting that the mitochondria play an important part in yolk-formation. A close relationship between the mito- chondria and yolk-formation has been recorded in the eggs of various animals by recent workers (Harvey, 1926; King, 1926; and Gardiner, 1917, and others). 196 IRENE F. HILTON GENERAL ACCOUNT. The ovary is single and median and is situated dorsal to the alimentary canal. It extends about two-thirds of the entire length of the thorax and tapers to a blunt point at its posterior end. Prom the anterior end of the ovary paired oviducts arise and run forward as wide thin walled tubes. These lie parallel to each other, dorsal to the alimentary canal after leaving the ovary, but at the anterior end each oviduct bends ventrally and later- ally from its fellow and runs posteriorly, ventral to the alimen- tary canal. The two oviducts open together on the first ab- dominal segment close to the opening of the spermathecae. It appears probable that fertilization takes place as the eggs leave the oviducal opening. Three distinct zones can be observed in the ovary; (Text- fig.l). (1) A multiplication zone situated in the posterior part of the ovary and consisting of small cells—oogonia—under- going mitosis. One or two cells at the tip of the ovary are larger than the other cells of the multiplication zone, these are probably primordial germ cells. (2) A narrow zone in which many of the cells show leptotene, pachytene, and synapsis stages in division. (3) A broad zone which occupies the whole of the anterior end of the ovary and which contains oocytes in a progressive series of growth phases. The anterior end of the ovary passes almost imperceptibly into the oviducts in which the later growth phases of the oocytes take place. Multiplication Zone. The oogonia at the posterior end of the ovary are very closely packed. Two or three nucleoli are present in the resting stage of the oogonial nucleus (figs. 1 and 2, PL 9). Owing to some difficulty which was experienced in obtaining good fixation of this part of the ovary the nature of the oogonial nucleoli is not perfectly clear. It was observed that one nucleolus is usually conspicuously larger than the others. After fixation by chrome- osmium technique followed by staining with iron haematoxylin, OOGENBSIS OF CALANUS 197 TEXT-FIG. 1. Diagram of longitudinal section through thorax showing zones of ovary, and arrangement of oocytes in the oviducts, m, multiplica- tion zone; s, synapsis zone; g, growth zone; a, half-grown oocytes; 6, older oocytes approaching maturation. NO. 294 O 198 IRENE F. HILTON this larger nucleolus stains rather more lightly than the smaller ones. After Champy-Kull technique it stains a uniform amber colour and after Mann's methyl blue eosin, bright pink. It is a plasmosome and remains spherical in shape. The smaller nucleoli are irregular in outline and stain more deeply with iron haematoxylin. With Feulgen's ' Nuclealfarbung' method these nucleoli give a deep pink reaction: they are karyosomes. The final oogonial mitoses, which actually mark the initiation of the growth phases of the oocyte, are visible in fixed and stained preparations immediately behind the synapsis zone of the ovary. The nucleoli disappear, or at least lose their staining properties, before the breakdown of the nuclear membrane: the chromatin network resolves itself into a very fine spireme thread which assumes a marked spiral arrangement within the nucleus. This thread breaks up into a number of long twisted chromosomes which shorten and thicken to form small rods. After the dis- appearance of the nuclear membrane these chromosomes, of which there appear to be about thirty-four, arrange themselves upon the equator of the spindle; metaphase, anaphase, and telophase follow in rapid succession. The chromosomes of this final oogonial mitosis swell in the telophase, forming a deeply staining mass at each pole of the spindle. The details of the events immediately following are rather obscured by this pro- cess, but the chromatin in the daughter cells formed by the division passes into the resting stage. Synapsis Zone. The resting stage of the nucleus of the early oocyte is of very short duration. Soon after the formation of the oocytes the nuclear network aggregates into masses which finally become drawn out to form a thread similar in appearance to the spireme thread of the early prophase nucleus of the oogonia. This thread breaks up into a large number of parts which become twisted and are extremely difficult to count in consequence. The chromatin then contracts to one pole of the nucleus and there forms a tangled, deeply staining knot. From this knot thick looped threads are seen projecting into the centre of the nucleus. Although no doubling of the threads was observed in the OOGENESIS OF CALANUS 199 leptotene stage, presumably this knot represents a synizesis figure in which the formation of bivalent chromosomes is taking place (fig. 4, PI. 9).