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Home , Fertilisation, Gametogenesis

GA.METOGENESIS A.NO FK UTILISATION IN NlCMATUS MUESLI. 101

Gametogenesis and in Nematus ribesii.

Li. Doncaster, M.A., Late Mackinnon Student of the Royal Society; Lecturer in Zoology in the University of Birmingham.

With Plate 8.

IN a previous paper1 I gave an account of the maturation and behaviour of the polar nuclei in several species of sawflies which develop parthenogenetically. In all these species there were two maturation divisions, giving rise to an nucleus and three polar nuclei, and in some cases fusion took place between the second polar nucleus and the inner half of the first. The egg nucleus sank into the and began to divide to form the , while the polar nuclei in all cases ultimately disintegrated. Since whenever the were clearly visible their number appeared to be eight, both in the maturation mitoses and in the later divisions in body-cells, it was concluded that no reduction in the ordinary sense took place. But if fertilisation ever takes place by conjugation of male and pronuclei, an obvious difficulty arises with regard to the number in fertilised , and since the process of fertilisation had not been thoroughly examined at the time when the paper referred to was written, it was necessary to leave the question open in the hope of finding a satisfactory answer later. This paper gives an account of 1 'Quart Journ. Micr. Sci.,' vol. 49, 1906, p. 561. 102 L. DONOASTER. the work done on the fertilised egg in Nematus ribesii and on the in that and other species. The methods used were generally the same as before, but it was found that, in searching for male pronuclei in the eggs of impregnated , thionin or gentian violet were more satisfactory stains than iron hEematoxylin, since they stain nucleus and cytoplasm but leave the yolk uncoloured. In the work on and the development of the ovarian egg, osmic fixatives (e. g. Flemming's fluid) were largely used in addition to sublimate.

THE FERTILISED EGG IN N. RIBESII. In some animals, e.g. the , the fertilised egg is easily distinguished from the virgin by the presence of asters in the yolk, but in the sawflies nothing of the kind can be found, and over 200 eggs had to be cut and examined before it became certain that conjugation of male and female pronuclei takes place. In very young eggs I had occasionally found minute rod-like bodies in the peripheral protoplasm near the anterior end, which are probably the heads of spermatozoa, and in somewhat later eggs bodies which appeared to be degenerating nuclei sometimes appear in a similar position. In eggs laid by impregnated females there are frequently in the yolk in front of the polar region more or less numerous small radiating patches of protoplasm which sometimes appear to contain indistinct nuclei, but protoplasmic masses not certainly distinguishable from these are found also in virgin eggs, although with less regularity. In eggs which are probably fertilised there are also fre- quently lines of protoplasm running inward from the edge of the egg near the point where the spermatozoa had been found. But in no case have I been able to recognise with complete certainty the male before the maturation divisions of the egg are completed, and after that stage nuclei found in the yolk may always be derived from the OAMETOGENESJS AND FERTILISATION IN NEMATUS RJBESII. 103 egg-nucleus itself. It is never possible, therefore, to say with certainty that a given egg is fertilised or not. But after much time spent in vainly trying to follow the entrance of the aud its conversion into the inale pronucleus, I at last was able to observe the conjugation of the sperm-nucleus with that of the egg, and so to prove that true fertilisation does take place (fig. 1). It occurs immediately after the maturation divisions; the three polar nuclei lie near the edge of the egg (two of them in the same section as the egg and sperm nuclei), and the fusion of the two inner polar nuclei has not yet taken place. The male and female pronuclei are in contact, the male being distinctly smaller than the female, but in another egg in which the same stage is seen the two are of about equal size. The subsequent stages of the conjugation and division of the nucleus have not been observed, but the section represented in fig. 1 leaves no reasonable doubt that normal conjugation takes place. It therefore became necessary to reconsider my previons conclusions with regard to the number of chromosomes, since never more than eight have been found in either fertilised or virgin eggs. I was thus led to work out the spermatogenesis, and to the fresh work on the maturation divisions to be described later. In my previous paper I mentioned that the behaviour of the polar nuclei appeared to be slightly different in fertilised and in virgin eggs, and subsequent work has confirmed this. In the virgin egg of N. ribesii the two inner polar nuclei fuse and give rise to a group of chromosomes, which is generally clearly double, with eight in each half. The two halves of the group do not lie far apart, and commonly remain without much change for some time. But in the majority of eggs from impregnated females the chromosome groups derived from the two inner polar nuclei lie completely and sometimes widely separated, as if the conjugation between the nuclei had been much less complete than in virgin eggs (figs. 2, 3, and 4). Further, in virgin eggs the polar chromosomes usually do not divide, at least for some 104 L. DONOASTEB. time, but in fertilised eggs they frequently divide compara- tively early, giving groups containing as many as sixteen chromosomes rather irregularly arranged in the " polar protoplasm." That this difference in behaviour is really connected with fertilisation is made probable by the fact that it rarely, if ever, occurs in eggs which are certainly virgin, but in the eggs laid by impregnated females it is frequent. Further, in several eggs laid by impregnated females the polar nuclei follow the typical virgin arrange- ment, and in these the little rayed protoplasm masses in the yolk, characteristic of fertilised eggs, are absent; but other eggs laid by the same female have the fertilised type of polar chromosomes, and in these the rayed protoplasm patches are also present. It appears, therefore, that the fertilisation of the egg- nucleus, or the presence of spermatozoa in the egg, in some way influences the behaviour of the polar nuclei.

SPERMATOGENESIS. When it had been shown that normal fertilization could take place in N. ribesii, it became necessary to re-examine the maturation divisions in order to make certain about the chromosome number, which I asserted in the previous paper to be eight both in the maturation and in the mitoses, and also apparently in fertilized eggs. The matura- tion of the egg begins immediately after it is laid, so that it is very difficult to get good preparations of the early stages, and I therefore decided to examine the matter first iu the development of the spermatozoa. In very young male pupae, shortly after the larval skin is cast in the cocoon, the testes consist of compact groups of cells at the sides of the alimentary canal. These cells (spermatogonia) have relatively large nuclei containing a conspicuous nucleolus (plasmosome) and eight or about eight chromatin masses apparently attached to the nuclear mem- brane (fig. 5). Division figures are scarce, but when found GAMET0GENES1S AND FERTILISATION IN NEMATUS BIBESH. 105 they show clearly about eight rather large chromosomes in the equatorial plate, which split so that eight travel towards each (fig. 6 a, b). At a later stage the testis becomes larger, and consists of lobes or compartments in each of which all the cells are in about the same stage. By the time the colours of the mature fly are beginning to appear the testis contains nothing bnt and nearly mature spermatozoa, but when the pupa is still white all stages from spermatogonia to spermatids are found in dif- ferent lobes, often in the same section. In the nucleus before the first maturation divisions the chromatin consists of a number of irregular masses (appa- rently about eight, but they are always rather indistinct). Shortly afterwards it becomes condensed into four more concentrated masses, each of which frequently appears double or quadruple (fig. 7 a,b, c). A spindle is then formed, and the four chromatic masses become tightly packed together in the equatorial plate, which is much smaller than in the sperniatogonial divisions. There are conspicuous centro- somes. The chromosomes in the spindle are so tightly packed together that it is difficult to be certain of their number, but a comparison of many mitoses leaves little doubt that there are four, each of which is bivalent (6g. 8 a, b). The appears to be of the heterotype form, resembliug the figures found by Moore in the cockroach 1 except that the chromo- somes are fewer and very much smaller (fig. 9 ft, b). They are, however, appreciably larger than the chromosomes in the maturation mitoses of the egg. The second maturation division is easily distinguished from the first by the fact that the spindle is of about half the diameter; the chromosomes are usually even more tightly packed, so as frequently to appear as a siDgle body, but in clearer cases there is little doubt that there are four (fig. 10 a, b, c). At the telophase a vesicular nucleus is formed, with the chromatin arranged round the edge

1 ' Quart. Journ. Micr. Sci.,' vol. 48, 1905, pp. 489 and 571, 106 L. DONCASTJill?. giving it a characteristic appearance (6g. 11). This becomes converted into the head of the spermatozoon. It must be concluded therefore that in the male the normal somatic number of chromosomes is eight; that four " gemini " appear iu the prophase of the first maturation division, and that finally four chromosomes are distributed by heterotype and hoinotype divisions to each spermatid nucleus. There is no trace of the " " formation described by Meves in the spermatogenesis of the bee.3

OOGENESIS. Iu the larva before it casts its skin within the cocoon the are much like the testes of the male, but larger, with bigger nuclei. The is enclosed in a cellular sheath, and some ovarian cells are already larger thau others ; these will form the eggs, while the more numerous smaller cells give rise to the nutritive and probably to the follicle-cells. All the nuclei at this stage contain about eight chromatin masses and one to three nucleoli (fig. 12). In the young pupa the egg tubes are already differentiated, and in a longitudinal section of a tube the changes in the nucleus can easily be followed. At the apex of the tube the nuclei are like those in the larval ovary; below this zone the chromatin becomes distributed through the nucleus as fine dots, which are often aggregated together in one part, as in a sort of synapsis (fig. 13). The egg nucleus then enlarges consider- ably, and the chromatin appears as an irregular thread; at this stage two or three nucleoli are generally conspicuous (fig. 14). After this stage yolk begins to be deposited, and before the egg is ripe the nucleus, which has been very large, dwindles so that in nearly ripe eggs I have been totally unable to find it. In the larval ovary mitoses may be found in the ovarian cells and in the sheath; those actually in the ovary appear to have eight chromosomes (fig. 15 a, b). But in the sheath in 3 ' Anat. Anzeiger,' xxiv, 1903, p. 29. GAMETOGENESIS AND FERTILISATION IN NEMATDS El BESII. 107 all the mitoses observed the number is more than eight; usually it seems to be sixteen, but in some cases the figure suggests more than sixteen very small chromosomes (fig. 16 a—/). Wilson1 has described spindles with double the somatic number in the ovary-sheath of Hemiptera, and regards them as abnormal,but the figures seen in N. ribesii certainly suggest that the eight chromosomes in the primi- tive germ-cells are compound, composed of a greater number of smaller units, possibly more than sixteen. In the pupal ovary the egg-cells are already definitely formed, and do not divide further, but merely undei'go the usual growth with, deposition of yolk. The follicle cells are now quite small, and an occasional mitotic figure is visible; these are rarely clearly defined, but appear to have eight chromosomes. When the egg has reached its full size the follicle cells become degenerate, with obscure dark-staining nuclei. Groups of similar degenerating cells are found here and there in the larval ovary. The fact that the chromosome number in the ovary, and probably in the follicle cells, is smaller than that found in the sheath is of considerable interest. In addition to the case described by Wilson, and referred to above, the same kind of thing has been observed by Petrunkewitsch in the bee,s in which he found the unreduced number to be sixteen in the egg, but sixty-four in the blasto- derm, and it is still more conspicuous in Ascaris, which, according to Boveri,has a large number of very small chromo- somes in the somatic cells, but only four in all the cells on the "germ-track," from the fertilised egg up to the maturation divisions of the germ cells.3 These facts suggest that it may happen not infrequently that the chromosomes in cells of the germ-track may be compound, and consist of a number of smaller units which become separated in somatic cells. But 1 " Studies on Chromosomes," iii, Mourn. Exp. Zoo.,' vol. iii, No 1, 1906. 3 'Zool. Jahrb.,' vol. xiv, 1901, Anat. und Onto;*., p. 573. ' Boveri, 'Ergebnisse fiber die Konstitution der Chromatisclien Substanz des Zellkerns.' (Fischer, Jena.) 108 J,. DONOASTELJ. even if this is found to be a phenomenon of general occurrence it does not necessarily affect the hypothesis of the individuality of the chromosomes in any essential point.

CHROMOSOMES IN THE MATURATION DIVISIONS OF THE EGG.

It has now been shown that in the spermatogonial and oogonial divisions there are eight chromosomes, and that in the these are reduced to four in the normal heterotype manner. These facts led me to re-investigate the maturation divisions of the egg, since in my previous paper (loc. cit.) I gave evidence that in both first and second polar mitoses the number was eight. The chromosomes in the maturation of the egg are much less easy to observe than in the spermatogenesis, for there are difficulties of technique to be overcome, and the egg has to be preserved at exactly the right moment. But after cutting some hundreds of eggs I have been able to convince myself that while there are two types of maturation. In some eggs no reduction takes place, and eight chromosomes pass into each of the four nuclei produced by the polar mitosis. In other eggs four double chromosomes are found in the equatorial plate of the second maturation division, and these separate into their component halves sending four into each daughter-nucleus (figs. 17—21). I have never obtained a sectiou of the first polar mitosis in which it is quite certain that there are four " gemini," although some figures strongly suggest this ; but at the close of the first division, when the chromosomes are arranging themselves to form the equatorial plate of the secoud mitosis, four double chromosomes are sometimes clearly visible (figs. 20, 21). I have also several preparations which show only four when the second polar mitosis is already begun. A comparison of figs. 17 and 19, 18 and 20, respectively, will show the difference between the reducing and equational types of maturation. It must therefore be concluded that in some eggs pairing GAMETOGENESIS AND FERTILISATION IN NEMATUS E1BESII. 109 (synapsis) of chromosomes takes place before the maturation divisions; resulting in the separation of complete chromosomes at one of the mitoses, while in other eggs no pairing takes place, and each chromosome undergoes two equational divi- sions. In connection with this it is noticeable that in the eggs having the equational type the eight chromosomes are about half the size of the four seen in reduced eggs. I have found the reduced type in eggs from both virgin and impregnated females, so that the view which first sug- gested itself, viz. that reduction only takes place in eggs which contaiu spermatozoa, is not tenable. Re-examination of my sections of Pcecilosorna luteolum confirms me in the belief that in that species, which yields females from virgin eggs, and is normally not fertilised, there are two equational divisions in all the eggs of which I have suitable preparations. In the developing egg the somatic mitoses of fertilised eggs appear always to have eight chromosomes; a larger number has never been found. This is what would be expected if only eggs which undergo reduction are capable of fertilisation. Tn virgin eggs commonly eight are found, but in some cases the equatorial plate seems to have four only, showing that reduced eggs when not fertilised can develop as far as the blastoderm stage (fig. 22). The number of eggs which die before hatching varies, in some batches beiug very small, in others more considerable ; it is possible that the reduced eggs are those which fail to develop to larvse. Since, however, it has been shown by the mitoses iu tbe ovary sheath that the chromosomes are possibly compound, it may happen that reduced eggs which are not fertilised restore the normal number of chromosomes by division of the compound chro- mosomes, as was asserted by Petrunkewitsch (loc. cit.) with regard to the bee.

The conclusion that the eggs of one species may either undergo reduction, or may retain the full number of chromo- 110 I.. DONCASTER-. somes, although in each case there are two polar mitoses, is of considerable interest. I know of nothing quite parallel with it hitherto observed in animals, but I think it not unlikely that in the two generations of the Gallflies, one of which is bisexual and the other purely female, a similar state of things may be found to exist. That there may be two types of egg, one of which is reduced and requires fertilisa- tion, and the other not reduced and parthenogenetic, is of course not infrequent, but in such cases the eggs generally have obvious external differences, and the unreduced form has only one polar body. A condition more nearly resem- bling that found in N. ribesii has been observed by Eosen- berg in Hieracium,1 in which the egg- in some on a head is reduced and can be fertilised, in others on the same head not reduced and parthenogenetic. But here again the number of maturation divisions is probably not the same in the two cases. In the bee, according to Petrunkewitsch, all the eggs ai-e reduced, but if not fertilised, the somatic number of chromosomes is restored automatically. The conclusions here reached may make it necessary to reconsider the provisional hypothesis of -segregation sketched in my previous paper, but until further facts are obtained in other species it seems premature to discuss the bearing of my results on the problem of the determination of sex. I have not found it possible, owing1 to the minute size of the chromosomes, to determine whether anything comparable with Wilson's " heterotropic " chromosome exists in Nematus. In some figures (e. g. the group represented in tig. 18) only seven chromosomes are visible instead of eight, but when they are so minute it is always possible that two are superposed and not distinguishable apart. Iu conclusion I take this opportunity of expressing my gratitude to Mr. J. E. S. Moore for allowing me to compare some of my preparations with his, and for valuable help in elucidating my sections. [NOTE.—In a series of eggs all laid by one on one 1 Brit. Ass., York, 1906. Discussion ou Fertilisation, Seels. D and K. GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBES1I. Ill day the polar mitoses are abnormal. The most extreme case (fig. 23) shows the "polar protoplasm," full of dots arranged roughly iu lines like iron-filings in a magnetic field. At each pole of the figure is a group of more conspicuous stained bodies which may be chromosomes. Some of the other eggs show a somewhat similar appearance on a smaller scale/and in others nothiug is clearly distinguishable in the polar protoplasm. In all the eggs the peripheral protoplasm is narrower than usual, and in the most markedly abnormal eggs it is practically absent. I have occasionally found appearances of the same kind, but much less pronounced, in eggs laid by other , but have not sufficient cases to be able to throw any light on their meaning.]

SUMMARY.

1. True fertilisation (conjugation of male and female pro-nuclei) may take place in N. ribesii, and the behaviour o£ the polar nuclei is slightly different in fertilised and virgin eggs. 2. In the spermatogenesis there are eight chromosomes in spermatogonial divisions; four " gemini" appear at the beginning of the maiotic phase, and by heterotype and homo- type mitoses distribute four chromosomes to each spermatid. 3. In the eight chromosomes appear in oogonial mitoses, but in divisions of nuclei in the ovary sheath more than eight are found, suggesting that the chromosomes of the germ-cells are compound. 4. In the polar mitoses of the egg two types of maturation are found. In some eggs there are successive equation divisions so that the egg nucleus and each of the three polar nuclei contains eight chromosomes. In other eggs normal reduction takes place, separating entire chromosomes from one another, and only four are found in each of the daughter nuclei. 5. It is probable that only such reduced eggs are capable 112 L. DONCASTBE. of fertilisation, but when unfertilised they may coutiuue to develop at least as far as the blastoderm stage. Birmingham University; November, 1906.

EXPLANATION OF PLATE. 8,

Illustrating Mi". L. Doncaster's paper on " Gametogenesis and Fertilisation "in Nematus ribesii."

All figures are drawn with an oil-immersion lens, but are not exactly on the same scale. Those illustrating spermatogenesis are more highly magnified than the remainder. All represent Nematus ribesii except figs. 12, 13, 14. ]?IG. 1.—Conjugation of male and female pronuclei. Three polar nuclei near the edge of the egg. FIGS. 2, 3, 4.—"Polar protoplasm" of fertilised eggs showing chromo- some groups derived from polar nuclei. FIG. 5.—Nucleus of . FIG. G.—Spermatogouial mitoses, (A) Metaphase, side view; (B) Equ- torial plate. FIG. 7, A, J>, C.—: three propliases of heterotype mitosis. (A) Showing 8 chromosomes; (B and c) Pairing to form & double chromatin masses. FIG. 8.—Heterotype mitosis, equatorial plate, (A) Pole view; (u) Side view. FIG. 9, A, B.—Heterotype anaphases. FIG. 10.—Homotype. (A) Pole view of equatorial plate; (B, c) Anaphase, side view. FIG. 11.—Spermatid. FIG. 12.—Young , N. lacteus. FIGS. 13, ]*.—Stages of growth of oogonium, N. lacteus pupa. FIG. 15.—Oogonial mitoses, larval ovary, (A) Pole view; (B) Side view. FIG. 16, A.—r.—Mitosis in ovary sheath witli more than 8 chromsomes. (A) Equatorial plate, pole view; (B, C) Similar stage seen from side and obliquely; (D, £, r) Anaphases. GAMET0GENES1SAND FERTILISATION ]N NEMA'l'US RIBESII. 113

FIG. 17.—Second polar mitoses, equational type, with 8 chromosomes. FIG. 18.—Second polar mitosis, equatorial plate in pole view, with 7 chro- mosomes, some preparing to divide. FIG. 19.—Second polar mitoses, metaphase; reduced type, with 4 chro- mosomes. FIG. 20.—Equatorial plate of reduced type, showing 4 double chromo- somes. FIG. 21.—Stage between first, and second maturation divisions, reduced type, with 4 double chromosomes each end. FIG. 22.—Two blastoderm mitoses, each witli 4 chromosomes and con- spicuous . FIG. 23.—Abnormal polar mitosis.

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GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBESII.