The Roles of the Nurse-cells, Oocytes and Follicle- cells in

The Roles of the Nurse-cells, Oocytes and Follicle-cells in Tenthredinid Oogenesis.

By

Professor A. D. Peacock, D.Sc, F.R.S.E.,and

R. A. R. Gresson, B.Sc.From the Department of Natural History, University College

(University of St. Andrews), Dundee.

With Plates 40-2 and 1 Text-figure.

CONTENTS. .PAGE

I . I N T R O D U C T I O N . . . . . . . . . 5 4 1

I I . H Y M E N O P T E R O U S O O G E N E S I S . . . . . . 5 4 2

I I I . M A T E R I A L A N D M E T H O D S . . . . . . . 5 4 4

I V . ^ P H E N O M E N A A S S O C I A T E D W I T H T H E N O U R I S H M E N T O F T H E

S A W F L Y E G G . . . . . . . . 5 4 5

1 . N u r s e - c e l l P r o c e s s e s . . . . . . . 5 4 52 . O o o y t e N u c l e o l a r B u d d i n g . . . . . . 5 4 93 . N u r s e - c e l l N u c l e a r C h a n g e s . . . . . 5 5 04 . T h e S e c o n d a r y o r A c c e s s o r y N u c l e i a n d t h e i r O r i g i n . 5 5 15 . C y t o p l a s m i c F l o w . . . . . . . 5 5 26 . F o l l i c l e - c e l l s 5 5 27 . E n g u l f m e n t a n d A b s o r p t i o n o f N u r s e - c e l l N u c l e i . . 5 5 4

V . D I S C U S S I O N . . . . . . . . . 5 5 5

V I . S U M M A R Y A N D C O N C L U S I O N S . . . . . . 5 5 6

V I I , A C K N O W L E D G E M E N T S . . . . . . . 5 5 8

V I I I . R E F E R E N C E S . . . . . . . . . 5 5 8E X P L A N A T I O N O F P L A T E S . . . . . . 5 5 9

I. INTRODUCTION.

SINCE Buchner's important researches on the hymenopterous

egg (1) there does not appear to have been any study, until the

present one, which follows up that part of his work relating to

oogenesis in sawflies (Tenthredinidae). Buchner was interested

particularly in the hymenopterous accessory nuclei, and the

larger picture of sawfly oogenesis was left far from complete.

542 A. D. PEACOCK AND R. A. R. GEESSON

The presentation of this picture, in fair completeness from thecytological aspect, is the purpose of this paper, while the physio-chemical phenomena, it is hoped, will form the matter of a futurecontribution.

II. HYMENOPTEROUS OOGENESIS.

Before citing our findings it would be well to give a prefatoryand brief account of present knowledge on certain points ofhymenopterous oogenesis. In sawflies the primary oogonia inthe ovarioles of the developing ovaries give rise to specializedcells, the follicle-cells, oocytes, and nurse-cells, which becomearranged in ovarian tubes (ovarioles) in a manner well known ascharacteristic for hymenoptera (Text-fig. 1). At first the nutri-tive chambers of nurse-cells are larger than the oocyte chambers,but, later, a reversal in their relative size takes place. This isdue to the growth of the oocyte at the expense of the nurse-cells,which dimmish in size and number. In the later stages numerousyolk-globules fill the oocyte except for a clear space situatedimmediately behind the opening from the nutritive chamber,and, from its position and appearance, it would appear thatthis area owes its character to the inflow of granular matterfrom the nurse-cells.

Paulke (6), for the honey-bee, states that the nurse-cells atfirst secrete and discharge a nutritive substance into the oocytes,and, later, become wholly absorbed by the latter, such absorp-tion being evidenced by the presence of large masses of nurse-cell nuclei in the upper part of the mature oocytes. He believedthe oocyte and the nurse-cells of the adjoining chamber to bederived from a single primary oogonium which gave rise tofour cells, one of which became an oocyte while the other three,by consecutive divisions, produced forty-eight cells. Thus theabsorption of the nurse-cells by the oocyte reunited the forty-nine daughter-cells as a single cell, the egg. The final disappear-ance of the nurse-cells is regarded by Snodgrass (7) as being dueto their absorption into the oocytes, because, during the laterphases of oogenesis, the oocytes contain small, grain-filledglobules which appear to be fragments of nurse-cell nuclei. He

OOGENBSIS OF TENTHREDINIDAE 543

was, however, unable to corroborate Paulke's detection of nurse-cell nuclei in the upper part of the mature oocytes. Eegardingthe nourishment of the hymenopterous egg considerable atten-tion has been focused on the structures designated ' secondary 'or ' accessory ' nuclei, and Hegner (4), in treating of these intwo species of C a m p o n o t u s (Formicidae), summarizes theconclusions regarding their origin arrived at by other workers.Thus Blochmann was the first to note the occurrence ofsecondary nuclei in Hymenoptera, and they were later observedby Marshall and others. Marshall, though unable to determinedefinitely their origin and fate in P o l i s t e s (Vespidae), agreedwith Blochmann that they arose by budding from the germinalvesicle. He was unable, however, to find any stages, butsuggested that they may act upon the nurse-cell substance andso make it available for the oocyte. Korschelt, studying themin Bom bus (Apidae), concluded, as did Will and Ayres, thatthey were derived from epithelial cells. Gross, working onB o m b u s and other Hymenoptera, believed them to be truenuclei originating from epithelial cells situated among the nurse-cells. But Hegner adds that this cannot be the case in formslike C a m p o n o t u s , where they appear ' before a follicle andnurse-cells are acquired '. Henneguy observed their occurrencein the wasp and the honey-bee. In the latter they appear to bederived from the follicular epithelial cells, but in the wasp theyoccur around the germinal vesicle and disappear very early.Hegner failed to find them in the honey-bee oocyte. Loyez,from studies on four species of B o m b u s , two species ofV e s p a and one of X y l o c o p a , stated that they arose fromthe germinal vesicle, follicular epithelial cells and nurse-cells,not by budding,' mais resultent d'une coagulation de substancesvenues du dehors a l'etat fluide ou granuleux et modifiees parle cytoplasme de l'ceuf '. Hegner, summarizing his results forC a m p o n o t u s , says that their ' origin by budding from theoocyte nucleus, or by the immigration of epithelial cells, seemsimprobable. The conclusion is reached that the oocyte nucleusgives off materials into the cytoplasm which become enclosed bya membrane and develop into nuclear-like bodies '. He was,

NO. 284 N n

544 A. D. PEACOCK AND R. A. E. GRESSON

however, unable to determine their fate. Buchner (1), in saw-flies, noted their appearance first as safranophile droplets andsmall bodies especially numerous in the anterior part of the egg.They originated either in close relationship with the egg-nucleusor farther from it in the egg-plasma. They also had a nurse-cellorigin and flowed over into the egg. In some species both pro-cesses usually occurred contemporaneously. The existence ofearly stages in the interior of the egg did not support the sup-position that accessory nuclei arose from the follicle cells,though, in A l l a n t u s sp., he regarded it as probable that thegranules were derived from follicle-cell emissions. In Ten-t h r e d a mesomelas , however, the young stage accessorynuclei, ' the trophonuclei' of Buchner, do not derive from theegg-nucleus, as in other Hymenoptera, but from the nuclei ofthe nurse-cells. He also described a process of budding fromthe accessory nuclei, but Wilson (8, p. 347) regards the evidenceas inconclusive.

To summarize, it has been found that for the nourishmentof the hymenopterous oocyte several sources exist. The nurse-cells, early in oogenesis, secrete and discharge a nutritive pro-duct into the oocytes, and, later, become wholly absorbed bythe latter (Paulke, 6). Secondary or accessory nuclei have beenfound in several types of Hymenoptera, and their manner oforigin varies greatly in the different forms. Thus, in sawflies(Buchner, 1), they originate in relation with the oocyte nucleusor from the nurse-cell nuclei, while in Bomb us and otherforms there is evidence that they derive from the germinalvesicle or from the follicular epithelial cells. Buchner regardsit as probable, for A l l a n t u s sp., that they originate fromfollicle-cell emissions, while Hegner believes that, in Cam-p o n o t u s, they are derived from material given off into thecytoplasm from the oocyte nucleus.

III. MATERIAL AND METHODS.

Material was obtained from specimens of P r i s t i p h o r apad i L., T h r i n a x m i x t a Klug, P l a t y c a m p u s l u r i d i -v e n t r i s Fall., and A l l a n t u s (Emphytus) pa l l i pe s

OOGENESIS OF TBNTHBEDINIDAE 545

Spin. (Enslin, 2), species widely separated in classification. Inall cases, except that of Allantus pal l ipes , the followingprocedure was adopted. The specimens were ovariotomizedunder tap-water and the ovaries fixed immediately by Bouin'spicro-formal method. Sections 10//. in thickness were stainedwith iron haematoxylin. The Allantus pall ipes ovaries,similarly dissected, were fixed by Bensley's method1 andsections of 5/u were stained with acid fuchsin.

The Pr i s t iphora padi material was obtained in season1921 from a pupa which had spent 11-15 days in the cocoon.Subsequent observation showed that the specimen was moreadvanced in development than were the others employed.

The Thrinax mixta ovaries were taken on July 11, 1921,from a pupating female still within the larval skin. It had beenreared from a wild larva collected the month previous.

The P la tycampus lu r id iven t r i s gonads came froma specimen which had spun in October 1920 and had over-wintered until June 29, 1921, when it was dissected. Theseovaries were the least developed of all.

The Allantus pallipes material was obtained from twopupae belonging to a strain the last of which had pupated onJune 19, 1927. Dissection was made 13 days later, and thesegonads proved to be slightly more developed than those ofPla tycampus lu r id iven t r i s .

IV. PHENOMENA ASSOCIATED WITH THE NOURISHMENTOF THE SAWFLY EGG.

1. Nurse-cell Processes.—In the ovarioles of P la ty -campus lu r id iven t r i s the nuclei of some of the nurse-cellsof the first organized nutritive chamber (which appears to bethe fourth of a series of seven nurse-cell clumps) are covered,in many cases completely, by a cloud of large darkly staininggranules of chromatin (Text-fig. 1,2); further, part of the interioris occupied by large dark-staining masses of irregular shape(fig. 5, PI. 40). The appearances seen indicate that these granules

1 Carleton, H. M., ' Histological Technique ', p. 244. 1925. Oxf. Univ.Press.

N n 2


TEXT-FIG. 1.

LETTERING.

cf, cytoplasmic flow ;ct, cytoplasmic tidemark;cyn, common cytoplasmof nurse-cells ; chc, chro-matin cloud; fed, darkfollicle-cell; fe, follicularepithelium ; ncc, nurse-cell chamber; nan, nurse-cellnucleus; nco, chamber-oocyte connexion; oo,oocyte ; oon, oocyte nu-cleus ; y, yolk; yg, granu-lar yolk.

For description see op-posite page.

OOGENESIS OF TENTHREDINIDAE 5 4 7

EXPLANATION OF TEXT-FIGURE 1.

Tenthredinid ovariole; schematizedlongitudinal section. 1. Proximal(anterior) region showing stages in the differentiation of oocytesand nurse-cells; anteriorly the immature cella are evenly dis-tributed and do not vary greatly in size, though one larger cellwould appear to be a potential oocyte; three oocytes are seenin successive stages of development, in the text the youngest beingdesignated the first, and those succeeding the second and thethird; anterior to the two largest oocytes the nurse-cells aregrouping to form nutritive chambers. 2. Nutritive chamber notyet separated completely from the anterior of the ovariole ; nurse-cells have increased in size but do not fill the chamber; eachnucleus in the anterior is surrounded by a chromatin cloud consist-ing of granules which have been given ofi from the nucleus into thecytoplasm ; in the interior of many of the nuclei the nucleolarmaterial is aggregating to form large masses. 3. First fully formedoocyte, separated by a membrane from the nutritive chamberanterior, and from that posterior, by a definite layer of immaturefollicle cells ; cells at the lateral margins form a thick folliclewall under a follicular epithelium ; the ooplasm is finely granularand evenly distributed. 4. Fully formed nutritive chamberseparated from adjacent oocytes ; nurse-cells have increased insize and completely fill the chamber ; a chromatin cloud occursaround each nucleus anterior in the chamber and the large intra-nuclear masses of chromatin have increased in size. 5. Fifth oocytesurrounded by follicle-cells except at the proximal pole where aplug of cytoplasm projects into the nutritive chamber anterior ;ooplasm finely granular and evenly distributed ; nucleolus bud-ding. 6. Sixth nutritive chamber ; chromatin cloud still investingcertain of the proximal cells ; the large intra-nuclear masses ofnucleolar material increased in size, and, in many cases, frag-menting ; the outline of some of the nurse-cells is becomingindistinct and some of the cytoplasm is flowing into the sixth oocytethrough a connexion established between the anterior oocytepole and the chamber. 7. Sixth oocyte showing connexion with thesixth nutritive chamber, and cytoplasmic flow ; the ooplasm hasbecome vesicular and contains large globules of yolk; fourfollicle-cells show dark tracts, that nearest the nucleus (rightside) giving ofi granular material, that in a corresponding posi-tion opposite projects into the oocyte. 8. Last (seventh) cham-ber ; nurse-cells completely broken up and most of the cytoplasmand nurse-cell nuclei and accessory nuclei have passed into thelast oocyte; three accessory nuclei (very small) and one nurse-cell nucleus lie at the entrance to the oocyte. 9. Last (seventh)oocyte showing engulfment and disintegration of nurse-cell nuclei;cytoplasmic flow well marked and shown as a light area extendingfrom the chamber-oocyte connexion into the anterior of the egg ;the nurse-cell nucleus nearest the oviduct nearly devoid ofchromatin and disintegrating ; numerous chromatin masses inthe ooplasm, many being derived from nurse-cell nuclei, others,most probably, from granular emissions of follicle-cells such as areshown in black.

548 A. D. PEACOCK AND R. A. R. GRESSON

of chromatin must originate from the nuclei. The latter areapparently undergoing a period of activity, as is evidenced bythe peculiar appearances of the irregular masses occupying theirinteriors. In the fifth nutritive chamber similar conditions exist,but are more pronounced in the smaller, proximal cells whichare the farther removed from the oocyte. Again, in the sixthchamber, similar appearances are in evidence except that someof the extra-nuclear granules occur a short distance from thenuclei while the internal masses are slightly larger. Several ofthe nuclei in the upper and middle parts of the last nutritivechamber contain the same kind of irregular masses, but, inmost cases, smaller than those occurring in the chamber preced-ing. Granules occur closely investing most of the proximaland middle nuclei, and are also found in the cytoplasm ofthe nurse-cells. The significance of the latter is discussedlater (p. 551).

In a few cases cells situated at the proximal end of theovarioles and anterior to the first fully formed oocyte showdark granules investing the nucleus. These are nurse-cells inthe early stages of differentiation.

The further developed A l l a n t u s p a l l i p e s material showsthis chromatin cloud in all the chambers of the series, while inthe fifth, sixth, and seventh a few granules occur in the cyto-plasm a short distance from the nuclei. Large irregular massesof chromatin are present in the nurse-cell nuclei of the thirdto the seventh (last) chamber.

The more highly developed ovarioles of T h r i n a x m i x t ashow these granule-invested nuclei in some of the second-chamber cells. The granules are numerically greater in thethird, fourth, and fifth groups, but, usually, they are notnumerous in the sixth and are absent from the last. In thethird chamber the nuclei of a few cells contain large irregulardark-staining masses like those of P l a t y c a m p u s l u r i d i -v e n t r i s. These are larger and more numerous in the otherdistal groups except in the last, where they are usually confinedto a few cells. In most cases they are more numerous in theproximal cells of a chamber.

OOGENESIS OF TENTHRBDINIDAE 549

The ovarioles of Pr i s t iphora padi , the most highlydeveloped in our series, present a somewhat different appearance.The nurse-cell nuclei do not contain large masses of chromatin,but, in most cases, are filled with large roundish dark-staininggranules. Many of the nuclei, especially proximally in theovarioles, are still surrounded by a chromatin cloud, whilenumerous granules of various sizes occur in the cytoplasm ashort distance from the nuclei. The larger granules of the cyto-plasm are vesicular, and are probably formed by the aggregationof the smaller granules emitted from the nurse-cell nuclei. Astriking feature of the older nurse-cells is the later substitutionof a clear space surrounding the nucleus instead of the earlierinvestment of granules (fig. 8, PI. 40). In section the nuclei showa halo.

2. Oocyte Nucleolar Budding.—Some of the fifth,sixth, and last oocyte nucleoli of P la tycampus lur idi -ventr is were most striking because of their many buds ofvarying size, density, and vesicularity. In Thrinax mixta ,stages of this process were observed in the nuclei of the fourth,fifth, and sixth oocyte. The ovarioles of P r i s t iphora padidid not show this feature, but, in a few cases, the nucleus con-tained large dark-stained masses which may have had originfrom the nucleolus. Generally speaking, what appears to be anearly stage of nucleolar budding is shown in fig. 10, PI. 41 ;here the buds have not become enlarged or clearly marked offfrom the nucleolus. A later stage is seen in fig. 11, PI. 41, whereone of the buds has waxed and is clearly losing its nucleolarconnexion. (A peculiar feature of this oocyte is that the nucleusis not clearly marked off from the cytoplasm at one point, but weare unable to attach any special significance to this.) Later still,free buds move away from the nucleolus (figs. 12 and 13, PI. 41),and, in an oocyte of Thrinax mixta , they were observedclose to the inner surface of the nuclear membrane. Furtherstages could not be traced in the ovarioles of P la tycampuslur id iven t r i s or Thrinax mix ta . Examination ofAllantus pall ipes material, however, although it did notshow budding stages, revealed, in many of the oocytes, rounded

550 A. D. PEACOCK AND E. A. R. GBBSSON

bodies of varying sizes staining in like manner to the nucleolus,and which, in all probability, originated from it. Many of thesebuds are closely applied to the inner side of the nuclear mem-brane, while similar bodies occur in close contact with its outersurface (fig. 15, PI. 41), or are situated in the egg substance somedistance from the nucleus. In a few cases some of these budsappeared as if they were passing through the nuclear mem-brane, and, occasionally, that part of the membrane immedi-ately overlying a bud was bulged or irregular in outline. Asthese buds are present on both sides of the membrane they mustpass through it. Although the actual process of budding wasnot observed in this material, the staining of these roundedbodies leaves no doubt of their origin from nucleolar buds.This is also borne out by the facts that some older ovarioles ofA l l a n t u s p a l l i p e s revealed no nucleolar budding, and thatthe rounded bodies were less numerous than in the youngerOvarioles. Thus it would appear that nucleolar budding takesplace at a much earlier stage of ovariole development inA l l a n t u s p a l l i p e s than in our other species. Unfortunatelythe material does not show the ultimate fate of these buds inthe ooplasm.

8. Nurse-cell Nuclear Changes.—Among the irregu-lar masses of a nurse-cell nucleus in a last nutritive chamberof P l a t y c a m p u s l u r i d i v e n t r i s , there were appearanceswhich suggested nucleolar budding such as already describedfor the oocytes. This cell (fig. 6, PL 40) is much smaller than itsneighbours and its nucleus is partly invested by chromatinand contains two large dark-staining bodies, the lower of whichappears to be a nucleolus giving off buds. In T h r i n a xm i x t a many of these irregular masses of the fifth and sixthchambers also seem to be budding (fig. 7, PI. 40). Again, in thelast chamber of some specimens, the ' buds ' look more distinctand ready to separate. Frequently ' buds ' are given off frommore than one mass in the same nucleus.

This process, however, is not nucleolar budding. Its realnature is revealed by the conditions seen in A l l a n t u s p a l -l i p e s . Here, in the first nutritive chamber, the nuclear

OOGENESIS OF TENTHREDINIDAE 551

material is aggregated into a compact mass (fig. 2, PI.40), while,in the second chamber, the masses become more irregular inoutline and appear about to break up. The third chamber showsa later stage in which small portions of material leave themain mass to be distributed throughout the nucleus (fig. 3,PI. 40). In the fifth and remaining chambers all the chromatinmaterial is broken up into irregular masses (fig. 4, PI. 40), likethose already described for other species. These observationsfor A l l a n t u s pa l l i pes evidently explain the nurse-cell' buds ' of other species as being merely late stages in frag-mentation of nuclear material.

4. The Seconda ry or Accessory Nucle i andthe i r Origin.—These bodies occur in the oocytes of Hyme-noptera and other insects during the later stages of oogenesisand finally disappear before maturation. Wilson (8) defines themas bodies which ' have the appearance of small nuclei, but whichdo not arise by division of the egg-nucleus '. They may begrouped about the germinal vesicle or be scattered throughoutthe ooplasm. They have been named ' secondary ' or ' acces-sory ' nuclei by different authors, while Blochmann (Hegner, 4)calls them ' Nebenkerne ' and Loyez (Hegner, 4) ' pseudo-noyaux '.

In our four species of sawflies it has been shown that thechromatin granules arising from the nurse-cell nuclei pass outfrom these nuclei (in a manner not precisely ascertained) intothe cytoplasm. Here they become vesicular and develop intosecondary nuclei, the larger of which seem to be coalescencesof several vesicles. In P r i s t i p h o r a p'adi many stages intheir formation may be seen (fig. 8, PI. 40).

But secondary nuclei have also another origin, namely, fromoocyte nucleolar buds, which, as already noted in A l l a n t u spa l l ipes (p. 550), occur on both sides of the nuclear membrane.These buds vary in size (fig. 15, PI. 41), but, unlike the granulesproduced by the nurse-cell nuclei, they do not aggregate in thecytoplasm into larger masses.

Further, as presently to be described, accessory nuclei mayalso be derived from follicle-cells.

552 A. D. PEACOCK AND R. A. R. GBESSON

5. Oytoplasmic Flow.—During their early stages intheir chambers the nurse-cells are sharply marked off fromone another, but, later, their boundaries become indistinctand finally disappear (fig. 31, PL 42). In Pr i s t iphora padithe cells of the sixth chamber are not distinctly separatedand some of their cytoplasm, together with their containedaccessory nuclei, can be seen passing into the oocyte throughthe narrow chamber-oocyte connexion. In this species thepassage was not present between any chamber and oocyteearlier than the sixth, its position being marked by a pro-jection from the oocyte into the preceding chamber ; further,the channel was not in direct communication with the nurse-cells' cytoplasm.

The cytoplasmic flow is indicated by the appearance of thematerial leaving the chamber and entering the oocyte. A tide-mark shows strongly in preparations because the inflowingsubstance stains much more lightly than the ooplasm. As maybe expected, the flow ia more pronounced between the lastchamber and oocyte, for the nurse-cell boundaries disappearcompletely and leave the chamber merely a mass of cytoplasmcontaining a few nurse-cell nuclei (Text-fig. 1, 8 and 9).

The sixth oocyte contains large globules of yolk placedtowards the periphery and the distal pole, but, at the anteriorpole, a clear space, yolk-free and staining lightly, still bearsevidence to the cytoplasmic flow. In the last oocyte the yolkis not 'present as globules but is finely divided.

In Thrinax mixta the sixth and seventh (last) oocytecontains yolk-globules evenly distributed. Cytoplasmic flow isnot well marked although chamber-oocyte connexions havebeen established in a few cases and the stream through thechannel and in the upper part of the oocyte is visible.

6. Follicle-cells.—In the follicle of the last oocyte ofPr i s t iphora padi , and in those of the fifth, sixth, andseventh (last) of Thrinax mixta , somewhat strikingappearances are manifested, for a certain number of cells, incontrast with their fellows, show up as partially or completelydark-stained (figs. 16 and 17, PI. 41). Their phases are as follows:


At first the cell stains slightly darker than its neighbours andits cytoplasm becomes more granular. The darkening becomesprogressively more pronounced until practically the whole cellfills with granules. The granules may extend as far as the polenext the egg, and, at this stage, the nuclear granules may belarger than the cytoplasmic. Presently the whole nucleus looksas if filled with intensely dark contents, while the cytoplasmbetween nucleus and egg is usually the region most darklystained (figs. 18 and 19, PI. 41). Later, the nucleus disappears,and all the cytoplasmic tract becomes intensely dark, and, fromat least one observation, processes from it extend into theoocyte (fig. 20, PI. 41). In T h r i n a x m i x t a two sectionsrevealed three striking features, firstly, the passage of granulesfrom cell to oocyte, secondly, their investment by a vesicle, and,thirdly, their regression from their point of origin (figs. 21 and24, PI.41). In P r i s t i p h o r a p a d i numerous dark granules,each of which has become surrounded by a vesicle, occur in theooplasm close to the dark follicle-cells, and, in a few cases,appear to have streamed from the follicle-wall. Larger granulesoccur some distance from the cells, and, although their positionsuggests that they are aggregations of smaller follicular granules,complete proof of such is not forthcoming, because, from thetechnique employed, it is not possible to differentiate them fromsimilar masses of known derivation from nurse-cell nuclei.These vesicle-invested granules of follicular origin resembleclosely those accessory nuclei described by Buchner and our-selves as originating from nurse-cell nuclei. A very late stageis seen in fig. 22, PI. 41, where no nucleus is visible, and wheremost of the dark-staining material has passed from the cell.The staining indicates that the nuclei disintegrate.

In the earlier stages the nucleus is more granular than thecytoplasm and most probably, therefore, the granules are ofnuclear origin. The nuclear emissions issue in all directions intothe cytoplasm but proceed chiefly towards the oocyte into whichthey finally pass. This is evidenced by the fact that the moredarkly stained region of the cell lies between the nucleus andthe egg. In transverse sections of T h r i n a x m i x t a many


follicle-cells are stained darkly in like manner to those justdescribed, and, in many cases, the intercellular spaces also (seefig. 23, PI. 41). Whether this intercellular material passed intothe oocyte or not could not be ascertained.

Another follicular feature was presented in a last oocyte ofP r i s t i p h o r a p a d i . Owing to shrinkage a space appearedat the pole between the egg-yolk and the follicle-cells next theoviduct (figs. 25 and 26, PL 41), and, thereby, it was revealedthat these follicle-cells have not the length of the others, butare provided with irregular processes which evidently insinuatethemselves into the egg-yolk. The processes are clearly markedoff from the main body of the cell as the sections show a lineextending across their common bases. Their significance hasnot been determined.

7. E n g u l f m e n t and A b s o r p t i o n of Nur se - ce l lNuclei.—As already described, the nurse-cells in the sixthchamber of P r i s t i p h o r a pad i are not sharply marked offfrom one another, while some of the cytoplasm, and its containedaccessory nuclei, pass into the egg (Text-fig. 1, 6 and 7). In thelast chamber separate cells are no longer present, the nucleibeing surrounded by a common cytoplasm formed by thefusion of the nurse-cells. The cytoplasmic flow is well marked inthis chamber, being so great that the chamber is shrunken insize while part of the interior appears void. The fate of thenurse-chamber contents is shown in Text-fig. 1, 8 and 9, andfig. 31, PI. 42. All the nurse-cell nuclei, no longer confined todefinite cells, are swept, one by one, with the cytoplasmicstream into the egg, and some even reach the distal pole.During their course some of the nuclei give off dark-staininggranules like those produced in the chambers. On reaching thedistal confines of the egg, they usually contain less chromatin,and this residue may be gathered into large masses stainingdarkly. Next, the nuclear outline becomes indistinct, and thechromatin disperses into the ooplasm. The final stage of nucleardisintegration is shown in fig. 9, PI. 40, where a clear ovoid spaceis surrounded by dark granules. The fate of the loose chromatinhas not been followed.

OOGENESIS OF TENTHRBDINIDAB 555

V. DISCUSSION.

The manner of origin of the accessory nuclei evidently variesgreatly among the different forms of Hymenoptera, for theyhave been described by various investigators as arising from(1) the egg-nucleus, (2) the follicle-cells, (3) the nurse-cells, and(4) by the coagulation of material in the ooplasm. They varygreatly also in size, appearance, and position. For example,those figured by Hegner and Paulke are large and assimilate toand cluster around the oocyte nucleus, while, in sawflies, theyare small, and, so far, have never been found in such condition.Our results show that, in at least the four species examined byus, they certainly originate from the nurse-cell nuclei, and, withthe possible exception of P r i s t i p h o r a p a d i , from the egg-nuclei also. In this we agree with Buchner, who states that, inthe sawfly T e n t r e d o m e s o m e l a s , they arise solely fromthe nurse-cell nuclei, while in other species they originate fromthe oocyte nuclei or may arise contemporaneously from bothsources. We are unable, however, to confirm his statementsregarding a process of budding from the accessory nuclei.

Our observations on oocyte nucleolar budding appear to forma new contribution in the study of hymenopterous oogenesis ;for, although we were unable to determine the ultimate fateof these buds in the ooplasm, in the case of A l l a n t u s p a l -l ip e s we observed them in positions which suggested that theywere passing through the nuclear membrane. Further, theywere traced into the egg substance where they gave rise toaccessory nuclei. Although secondary nuclei have been describedas deriving from oocyte nuclei we do not know of any record oftheir origin from nucleolar buds.

For reasons already given the process involved in nurse-cellnuclear changes is very different from that of oocyte nucleolarbudding. It is really the breaking-up of masses of aggregatednuclear material, the fragments of which become distributedthroughout the nucleus. This conclusion appears to be new.

The manner by which the nurse-cells supply nutriment tothe developing oocytes closely parallels that described by Paulke

556 A. D. PEACOCK AND R. A. R. GRBSSON

and Snodgrass for the honey-bee. Thus, after the chamber-oocyte connexion has been fully established, some of the cyto-plasm, together with accessory nuclei, passes through it to theoocyte, while, finally, the residual cytoplasm and its containednuclei follow, and are absorbed by the egg. This observation hasnot been previously noted, and it is curious that it is not includedin Buchner's findings. Additional interest attaches to it, because,while Paulke describes such as occurring in the honey-bee, Snod-grass states his inability to confirm the former's finding.

The detailed description of the dark-staining tracts in thefollicle-cells and their emission of granules into the ooplasmconstitutes another new contribution to the subject, for,although Buchner regarded it as probable that the accessorynuclei of A l l a n t u s sp. arose from such emissions, we havenot been able to trace any records relating to it among theHymenoptera, or, indeed, among most insects. This dark-staining reaction seems to be indicated in one of Gross's draw-ings (3) on Hemipterous follicle-cells, but its significance is notdiscussed by him. Murray (5), working on the secretion of thefollicle-cells in G r y l l u s a b b r e v i a t u s and N e m o b i u sf a s c i a t u s (Orthoptera), describes granules in the follicle-cellsand concludes that they are intermediate stages between thelipoid granules in the follicle-cell nuclei and those surroundingthe yolk-globules in the egg. With our method of staining itwas not possible to determine the nature of the granules originat-ing from the darkly-staining follicle-cells, for, although manyresemble the smaller yolk-globules, others are strikingly similarto the accessory nuclei. Here, possibly, we have a third sourceof these bodies, the other two being the oocyte and nurse-cellnuclei.

VI. SUMMARY AND CONCLUSIONS.

1. Ovary formation in Tenthredinidae follows the generalhymenopterous plan.

2. Nurse-cell phenomena are as follows : the nuclei of thefirst nutritive chamber are surrounded by a chromatin cloudand many of them contain irregular darkly-staining masses of


nuclear material, which masses may also be present in the riperchambers ; the granules given off from the nuclei into thechromatin cloud eventually become surrounded by a vesicleand give rise to the ' secondary ' or ' accessory ' nuclei.

3. Oocyte nucleolar phenomena show the following: thenucleoli in T h r i n a x m i x t a and P l a t y c a m p u s l u r i d i -v e n t r i s give rise to buds which become free; in one case budswere observed close to the inner surface of the nuclear mem-brane ; in A l l a n t u s ( E m p h y t u s ) p a l l i p e s are shownwha,t are apparently later stages of this process, viz. the passageof the buds through the nuclear membrane into the egg sub-stance and their formation there into accessory nuclei.

4. The fate of the nurse-cells is shown in the older nutritivechambers and oocytes—the cell boundaries become indistinctand some of the cytoplasm, together with contained accessorynuclei, passes by a narrow channel into the oocyte. The cyto-plasmic flow becomes more marked in the last chamber. In thefinal stages, shown in the last chamber, all the cells lose theirboundaries and the common cytoplasm passes into the oocyte,carrying with it the free nuclei to their engulfment and absorp-tion in the ooplasm.

5. Some of the follicle-cells surrounding the last oocyte inP r i s t i p h o r a p a d i , and the fifth, sixth, and seventh ofT h r i n a x m i x t a , contain granular dark-staining materialwhich may completely fill the cell, these granules probablyoriginating from the nucleus. They pass out of the follicle-cellinto the egg where they become surrounded by vesicles, and,finally, present an appearance indistinguishable from that ofaccessory nuclei.

6. Secondary or accessory nuclei, therefore, have a threefoldorigin, namely, from the nuclei, of nurse-cells and oocytes andfrom follicle-cells, their source of derivation in the last beingthe follicular nuclei.

7. The follicle-cells of the distal pole of the last oocyte of oneovariole of P r i s t i p h o r a p a d i have processes which in-sinuate themselves into the ooplasm.

8. The phenomena of oogenesis described in these four species


of sawflies, while embracing certain which have not hitherto

been recorded, conform, in essentials, with those already dis-

covered for Hymenoptera generally.

VII. ACKNOWLEDGEMENTS.

This paper has been prepared from material collected during

studies in the parthenogenesis of sawflies, and, as grants for

such researches have been furnished by the Royal Society of

London and the British Association for the Advancement of

Science, the financial assistance of these bodies is gratefully

acknowledged.

VIII. REFERENCES.

1. Buchner, P.—" Die akzessorischen Kerne des Hymenoptereneies ",' Arch. f. Mikr. Anat.', Bd. 91. 1918.

2. Enslin.—" Die Tenthredinoidea Mitteleuropas", ' Deutsche Entom.Zeitschr.', Heft 7. 1912.

3. Gross, J.—" Untersuchungen iiber das Ovarium der Hemipteren,zugleich ein Beitrag zur Amitosenfrage ", ' Zeitschr. Wiss. Zool.',Bd. 69. 1901.

4. Hegner, R. W.—" Studies on Germ-cells. IV. Protoplasmic Differentia-tion in the Oocytes of certain Hymenoptera ", ' Journ. Morph.',vol. 26, no. 3. 1915.

5. Murray, M. R.—" Secretion in the Amitotic Cells of the Cricket EggFollicle ", ' Biol. Bull.', M.B.L. 50. 1926.

6. Paulke, W.—" Ueber die Differenzirung der Zellelemente im Ovariumder Bienenkonigin (Apis melliflca)", ' Zool. Jahrb., Anat. u. Ontog.',14. 1900-1.

7. Snodgrass, R. E.—' Anatomy and Physiology of the Honey-bee ',pp. 270-i. 1925. McGraw-Hill & Co., New York.

8. Wilson, E. B.—' The Cell in Development and Heredity', pp. 346-9.1925. Macmillan Co., New York.

OOGENESIS OF TENTHREDINIDAE . 559

EXPLANATION OF PLATES 40, 41, AND 42.

The drawings on Plates 40 and 41 were made by means of aZeiss camera lucida and a Watson ' Service ' Microscope. Forfigs. 8 and 9 a Eeichert l/12th objective was used, for figs. 24and 25 a Watson l/6th, and for all others a Eeichert l/10th. Theeye-piece in all cases was a Hawksley no. 4, x 10.

LETTERING.

anx accessory nucleus ; ch, remains of chromatin of nurse-cells ; chr,chromidia ; fc, follicle-cell; fed, dark follicle-cell; fcp, follicle-cell processand base ; fe, follicular epithelium; fs, follicular space ; nc, nurse-cell;ncn, nurse-cell nucleus ; ns, nucleolus ; nsb, nucleolar bud ; oo, oocyte ;oon, oocyte nucleus ; op, ooplasm ; sp, space ; y, yolk.

PLATE 40.

Figs. 5 and 6 from P l a t y c a m p u s l u r i d i v e n t r i s ; figs. 1, 2, 3,and 4 from A l l a n t u s ( E m p h y t u s ) p a l l i p e s ; fig. 7 from T h r i n a xm i x t a ; figs. 8 and 9 from P r i s t i p h o r a p a d i .

Fig. 1.—Early stages of nurse-cells anterior to the first fully formednutritive chamber.

Fig. 2.—Nurse-cells in first nutritive chamber. The chromatin material isaggregated into a compact mass.

Fig. 3.—From third nutritive chamber, showing chromatin passing outfrom the main mass.

Fig. 4.—Sixth nutritive chamber, showing a later stage in the fragmenta-tion of the nuclear material.

Fig. 5.—Nurse-cell from first complete nutritive chamber, showingnucleus covered by a chromatin cloud while part of the interior is occupiedby large irregular masses.

Fig. 6.—From last nutritive chamber, showing irregular masses in thenuclei; those in the smaller nucleus present an appearance such as resultsfrom oocyte nucleolar budding.

Fig. 7.—Nurse-cells showing a similar appearance to those in fig. 6.Fig. 8.—Nurse-cells more advanced in development than above and in

which the chromatin cloud has been replaced by a clear space ; accessorynuclei are seen scattered throughout the cytoplasm.

Fig. 9.—Showing the disintegration of nurse-cell nuclei which havepassed into the egg ; in the nucleus figured the remains of the chromatinare aggregated into large masses ; the final stage shows a clear space sur-rounded by granules.

NO. 284 O 0


PLATE 41.

Fig. 11 from P l a t y c a m p u s l u r i d i v e n t r i s ; fig. 15 from All an-t u s p a l l i p e s ; figs. 10, 12, 13, 21, 23, and 24 from T h r i n a x m i x t a ;figs. 14, 16, 17, 18, 19, 20, 22, 25, and 26 from P r i s t i p h o r a p a d i .

Kg. 10.—Early stage of nucleolar budding ; the buds are not clearlymarked off from the nucleolus of the oocyte.

Fig. 11.—Later stage : one of the buds is large and losing its nucleolarconnexion.

Figs. 12 and 13.—Free buds which have moved away from the nucleolus.Fig. 14.—Dark masses in oocyte nucleus which are probably of nucleolar

origin.Fig. 15.—Nucleolar buds in close contact with the inner surface of the

nuclear membrane ; one bud appears to have passed through the mem-brane and is in contact with the membrane's outer surface.

Figs. 16 and 17.—Early stages in the formation of granular tracts in thefollicle-cells ; in each figure one of the cells is seen to be more darklystained than its neighbours.

Figs. 18, 19, and 20.—Later stages : the cell becomes filled with, dark-staining material, while the cytoplasm between the nucleus and the egg isusually more darkly stained than elsewhere ; later, the nucleus disappearsand processes from the tract extend into the oocyte.

Fig. 21.—Late stage : granules are being passed out of the follicle-cellinto the oocyte, where many of them have become surrounded by vesicles.

Fig. 22.—Late stage : the nucleus has disappeared and most of thedarkly-staining material has passed out of the cell into the egg.

Fig. 23.—Follicle-cells at the side of the egg cut transversely and showingdarkly-stained intercellular tracts and darkly-stained cells.

Fig. 24.—Oocyte showing dark-staining follicle-cell from which granulesare being passed out into the oocyte, where they become surrounded by avesicle ; the large vesicular mass situated some distance from the folliclemay have been formed by the aggregation of granules originating in thesame way, while those granules close to the follicle wall at the opposite sidewere derived from another cell.

Figs. 25 and 26.—From a terminal oocyte, showing a space at the distalpole between the egg-yolk and the follicle-cells ; these follicle-cells are notso long as those laterally situated, but are provided with irregular processeswhich evidently insinuate themselves into the egg-yolk ; these processesare clearly marked off from the main bodies of the cells as the sections showa line extending across their bases.

OOGENBSIS OF TENTHRBDINIDAB 561

PLATE 42.

Photo-micrographs of sections of P r i s t i p h o r a p a d i ovariole. Forcontrast certain features such as nuclei and yolk have been touched up anddarkened with Chinese ink ; nothing has been added, but artifacts visiblein the mounting medium of the preparations have been eliminated.

Pig. 27.—Follicle-cell containing dark-staining material which occurschiefly between the nucleus and chorion. Nurse-cell nuclei which havepassed into the oocyte are also shown.

Fig. 28.—Later stage of follicle-cell, the dark tract extending from theouter pole ; no nucleus is visible.

Fig. 29.—Distal region of oocj7te showing dark stellate mass whichappears to be passing out of one of the follicle-cells, its true position beingdifficult to determine owing to the fact that many of the follicle-cells arecut transversely.

Fig. 30.—Nurse-cell nuclei in the anterior of an oocyte with two passingthrough the connexion from the nutritive chamber.

Fig. 31.—Last nutritive chamber and oocyte showing connexion throughwhich most of the nurse-cell nuclei have passed ; only a few nuclei remainin the chamber; the cell boundaries have disappeared, while most of thecytoplasm has passed into the oocyte leaving a clear space in the centre ofthe chamber ; the cytoplasmic flow through the connexion is well marked.

Fig. 32.—Last oocyte showing the space at the pole between the egg-yolk and the follicle-cells next the oviduct; nurse-cell nuclei are seen, thelower one containing large masses of chromatin.

0 0 2

Quart. Journ. Micr. Sci. Vol. 71, N. S.f PL 41

°P

Scale of all figs, except'0/mm. 24 and 25

I I I I I zzPeacock and Gresson.

Quart. Journ. Micr. Sci. Vol. 71, N. 8, PI. 42

_„__ 32

Peacock and Gresson.

The Roles of the Nurse-cells, Oocytes and Follicle- cells in

Documents

Transcript of The Roles of the Nurse-cells, Oocytes and Follicle- cells in