EGG FORlIATION I N A LEECH

HAROLD IIEATII Hopkins Marine Station of Stair f o , tl T;itacerszt!j

TWO PLATES (ELEVEPI’ FIGURES)

AUTHOR’S ABPTKACT

I n a leech infesting the Alaskan codfish germinal masses in the ovary proliferate sec- ondary groups, comprising about forty cells in which a follicle and central supporting cell early differentiate. Acti1.e division results in approximately 500 cells which apparently develop ductules extending to a point on tlie surface of the egg. Granules of unknown origin then appear in each nurse cell, and are drawn down the ductules into the egg which can now be distinguished. Reasons are giwn for the belief that the nutritive material is drained from the nurse cells hy amoeboid activity of the egg. I n early stages the nutritive material forms a loose reticulum which gradually becomes transformed into a more extensive network, persisting until the msturation divisions. Tn this latrst period the follicle and nurse cells, which beeorne shrunken as the ovum enlarges, usually are stripped off and soon disintegrate.

In 1913 Jorgensen (’13) published an account of the de- velopment of the egg of a leech (Piscicola), which, it will be remembered, belongs to Korschelt aiid Heider ’ s alimentary- riutrimentary class where the fmictional ovum gains its yolk supply as a result of the activity of several associated nurse cells. Recently, in connection with the study of germ cells in several groups of invertebrates, I have examined a score or more individuals of a species of leech infesting the bran- chid cavity of tlie hlaskaii codfish ( Gaclus macrocephalus) . In this species the egg development conforms in certain re- spects to the plan described by Jorgensen, yet departs rather widely from it in certain fundamental particulars, as the following account demonstrates.

The material was preserved in formaldehyde, corrosive- acetic, aiid Bouiii’s picro-acetic-formol mixture. Several stains were used, Delafield ’ s aiitl Heidenhain’s haematoxylin, occasionally with acid fuchsiii as a counterstain, proving the most satisfoctory.

While tlie earlier stages of germinal activity are not rep- reseiited in the present collection, it is probable that they

333

334 HAROLD HEATH

follow the type of development occurring in Piscicola. According to Jorgensen, repeated divisions of tlie elements of the germinal wall result in a mass of cells projecting into the ovarian cavity. These masses subsequently break loose, float freely in the egg sac, and then bud off small clumps in which egg and nurse cells become differentiated. In the present species each ovary contains one such floating cell group, more or less ellipsoidal in form, from one end of which the groups of oogonia are in process of formation and separa- tion (fig. 1). Furthermore, the cavity of each ovary contains myriads of small cells (a few are shown in fig. 1) which prac- tically fill all of the space between the developing eggs. From Jorgensen’s account it appears that these arise at tlie same time and from the same source as the egg-nurse cell groups. If such be their genesis in the present species, it occurs at a period prior to the one represented in figure 1, for, though they present the same appearance and are of the same size as those of the floating germinal masses, there is no indication that they spring from such a source. Throughout the de- velopmental stages of egg diff’erentiation these free isolated cells undergo mitotic division, but a t a later time, when the majority of the ova are in the initial stages of maturation and the clitellar glands are highly active, the nuclei of these floating cells become eccentrically located, and their cyto- plasm indicates glandular or degenerative changes, but in the absence of older material nothing is known of their fate.

The germinal masses which give rise to the secondary groups of oogonia are ellipsoidal in form and measure ap- proximately 0.225 mm. in greater diameter. They comprise multitudes of elements destined to become ova and nurse cells, among which arc relatively few cells of larger size, each of which becomes transformed into what may be termed a follicle cell (fig. 1, f). Judging from the material in hand, these germinal masses persist until a relatively late period, when the oldest ova, free in the ovarian cavity, have reached the stage represented in figure 2. As long as they exist, a few of the component cells are usually undergoing mitotic di- vision.

EGG FORMATIOX IN A LEECH 335

As noted previously, the ellipsoidal germ masses continu- ally are liberating groups of cells from one pole. These sec- ondary groups measure approximately 0.036 mm. and comprise about forty cells, one of which, the follicle, is already differentiated. At a slightly later stage, a central cell becomes distinguishable from the others, owing to its larger size and elongated more vesicular nucleus. It subsequently becomes differentiated into what may provisionally be termed the sup- porting cell of the nurse-cell group. The condition of affairs at this time is represented in figure 8, s.

At this point the follicle cell occupies a superficial position with its nucleus embedded in a relatively small mass of proto- plasm, while its more peripheral portions are fashioned into a membrane completely surrounding the remaining cells. In several abnormal groups, where the number of oogonia is not over eight or ten, these are completely embedded in the cytoplasm of the relatively large follicle cell. On a small scale this probably represents the normal state of affairs in early stages, the nutritive cells being actually embedded for a time in the substance of the follicle before this shifts to the periphery of the group. This implantation process occurs in the main germinal mass ; indeed, it appears to be responsible for the cutting off of these secondary groups of oogonia, but owing to the difficulty of tracing cell boundaries, the details of the process are unknown.

From this point, where each oogonial cluster consists of one follicle cell, one supporting cell, and about forty nurse cells, the main feature of further development f o r a consider- able period consists in the division of the nutritive cells. These multiply by mitosis until there are not less than 500, forming a compact group 0.124 mm. in diameter. Cell divi- sion beyond this stage apparently ceases, but growth proceeds at a steady rate, owing to the development of a canal system destined t o convey products from the nurse cells to the de- veloping ovum.

The stage represented in figure 6 is thus reached where the entire group measures approximately 0.26 mm. in diameter

R 3 G HAROLD HEATH

aiid tht. future ovum has become distinguishable. Beyond this poiiit there is practically no growth of the group as a whole, the average diameter of twenty mature ova being 0.279 mm. The growth of the egg, however, proceeds a t a steady ratch by a process which remains obscure in certain respects.

111 the stage represented in figure 7 the structure of tlie nurse cells is not entirely clear. It appears certain that all with the exception of the supporting cell have changed, after the period of cell multiplication, from more or less spherical to pyriform shape (fig. 4) . In rare instances the cell stalk lias been traced for a distance fully three times the diameter of the main ccll body; but if it actually does extend beyond this point, its tortuous course precludes the possibility of followiiig it in sections. However, I am of tlie opiniori that each ccll stem, in reality a clactule, does extend from the cell lood)7 to the ovum.

The central or supporting cell is ail uiisolvetl problem. I t s general appearance is represented in figure 5. Here the cyto- plasm in iiormal cases is so scanty in amouiit and the atlja- cent cells are so closely crowded against it that it appears to be a very restricted body. In rare, possihlp ahnormal, instances, however, its cytoplasm can he traced for a coii- siderable distance in various clirectioiis as it extends out- wardly hetm-eeii the adjacent iiiirse cells. In yet other cases, vertaiiily abnormal, it does extend between the cells. In tlie simplest example, represented in figure 9, the follicle cell is absent, aiid a few iiursc cells are attached to the surface of the supporting cell in whose substance a re embedded two additioiial iiurse cells. If the iiumher of tliese surrounded nutritive cells was increased to 500, i t is helievcd it would caorrectly i*epreseiit the relation of the supporting cell to the others. Under such (normal) c+wimstances, the nurse cells, or a t least their ductules, are thus covered with a delicate sheath, which may serve to hold them in a position to allow of their proper functioning after the manner described in a following paragraph. Certain it is that the large size of the supporting-cell nucleus indicates that it belongs to a cell body also of considerable bulk.

EGG FORMATION I N A LEECH 337

I n the stage immediately preceding the first risible differen- tiation of the fiinctional egg (shortly before the stage repre- sented in figure 6) granules appear in the cytoplasm of each nurse cell. Since the material is not especially favorable for the study of their origin, 110 attempt was made to discover whether they represent mitochondria, extruded iiucleolar ma- terial, or some other cell product. The position of this ma- terial in the cell varies, as may be seen in figure 4. Sooner or later it tends to form a mass in the neighborhood of the nucleus, and from this locatioii extends along the axis of the cell stem as far as this can he followed. Throughout the nurse-cell group, especially in the more central portions, there are scores of what appear to be cell stalks cut through at various angles and with axially arranged granules (figs. 4, 6, 7) . 111 other words, as iioted previously, it is believed that the tubular stalk of each niirse cell extends, and the granules are coiive;Ved, from the main cell h d y to a point on the sur- face of the developing egg cell.

Whether these <:ellular caiials remain independent of each other, or whether they unite before reaching the egg is uii- certain, though appearances suggest that there is a certain amount of fusion. In figures 2 and 7 two streams of granules are represented as entering the egg. Since this is tlie state of affairs in dozens of cases, it would appear that there is a minute circle of pores in the vitelline membrane rather tliaii a single opening. However this may be, it is certain that the egg membrane is perforated, and granules form a contiiiuous stream from near the point of entrS into the substance of the ovum. It may be added that in exceptional cases the pores are distributed over a much wider area than is represented in the figures.

I n figures 2 and 3, wliere the outline of the egg is accurately represented, it will be see11 that its free surface is irregular in contour, and indicates, I believe, tlie method whereby the granules from the nurse cells arc brought into the egg. Each elevation on the surface of the ovum is a pseudopodium. As it develops with the pushing outward of the egg substance a

338 HAROLD HEATE

mass of granules of corresponding bulk is drawi from the nurse cells into the egg. Other pseudopodia form with the same result. None are ever retracted, but new ones are built upon the old until the nurse cells are drained of tlieir entire product. In support of this opinion, the following evidence is offered. In twenty-two cases the nucleus of the follicle cell was located in contact with the egg proper (hence there is no definite polarity in the cell group, so far, at least, as the follicle is concerned), and in sixteen instances this produced a marked effect on the distribution of the granules within the egg. In eleven of these examples the granules lying between the point of entry into the ovum and the follicle nucleus were almost entirely absent, suggesting that the pres- ence of the follicle nucleus prevented the formation of a pseudopodium, and hence inhibited the withdrawal of gran- ules in its direction. In five other examples the granules formed a triangular mass with tlie base as broad as the folliclt. nucleus and adjacent to it, while the apex was located at the point of entry in the vitelline membrane. In this case the effect may be explained by assuming that a pseudopodium as broad as the follicular nucleus, and surmounted bv it, was formed, and as in other cases the width of the stream of granules is approximately proportional to the size of the psen- dopodium. In the six remaining cases no peculiarity of gran- ule arrangement could be discorered. Whether this was the result of a more than usual fluidity of the follicle or to a sub- sequent rearrangement of the egg contents is unknown.

During the early stages of egg development the granules tend to follow more or less curved planes, and in cross-section often present the appearance of definite membraiies (fig. 3 ) . In later stages this plate-like configuration is changed into a spongy reticulum which becomes a striking feature later in the development of the ovum (figs. 7 and 10). Finally, at the time the first maturation spindle has commenced to form, this network may yet be present o r in many instances it map be absent altogether due to the uniform distribution of the granules.

EGG FORMATION IN A LEECH 339

The granules in the nurse cells appear to be slightly smaller on the average than those distributed throughout the egg. Their staining reactions, however, are essentially the same and continue to remain so as far as their history has been followed. If they are or become yolk, as is entirely probable, they undergo very little visible change during their sojourn in the egg, at least up to the time of maturation.

As previously noted, the nurse-cell group gradually shrinks in size as the egg develops, there being very little increase in bulk of the entire mass throughout the entire period of egg development. In the oldest stages followed, where the first maturation spindle had formed, the follicular membrane and the nurse cells had entirely degenerated in some instances ; in other cases they continued to form a sheath about the egg (as in fig. 11).

Jorgensen calls attention to the significant fact that, after the division period of the oogonia, nurse cells and oocytes alike may pass through synaptic stages. I n the present species there is some evidence that a similar process occurs, though it may be merely a preparation for the usual division process which never extends beyond the spireme stage. Prac- tically all of the 500 cells advance to this point simultaneously as though under the sway of some very definite coordinating influence, and as uniformly return to a ‘resting’ condition from which they never emerge.

The details of the maturation of the ovum, so far as they have been determined, agree closely with what occurs in Pis- eicola. There is the same large nucleus, a similar paucity of chromatin, but no centrosome has been discovered until after the nucleolus has largely dissolved or immediately before the first maturation division. At that time from one to three centrosomes surrounded by conspicuous asters may be seen usually at some distance from each other and from the nu- cleus. Whether they play any part in the formation of the definitive spindle cannot be determined from the material in hand. Furthermore, in the oldest stages (fig. 11) there is usually a spiral twist to the central spindle.

340 HAROLII HEATH

In Piscicola, as described hy Jorgeiisen, the oogonia sepa- rate from the main germinal mass in groups of from six to eight, and b>- repeated divisions become increased to approxi- mately fifty c d s . During the process one, often two, and rarely three ova are produced, the entire group being sur- i+oundcd by two or three cells of larger size. One of these last-named elements becomes partially embedded in the re- maining nutritive cells, and tentatively was compared by .Jiirgensen to the so-called Verson or Spengel cell occupying a central position in the iiurse (.ell of Koiiellia, for example. This appears to he a correct interpretation, and I believe the comparison may he extended a step further to include the supporting cell in the present species, both cells being homol- ogous elemeiits.

The similarity of the two species is further heighteiiecl hy the fact that some of tlic nurse cells in Jorgeiiseri’s fig Oures (especially fig. 1) sliow well-developed stalks. Furthermore, the central portions of the yo~mg oogonial groups are de- scribed as coiitairiing a cavity possibly filled with some nu- trient fluid (figured apparently as a precipitation product clue to reagents). T n the present species a section to one side of the nucleus presents an appearance not unlike this. Tlic cavity, however, in every case is found in reality to con- sist of large iinmbers of ductules, and not a precipitatioii product. I suspect this will prove to be tlie case in Piscicola when carefully prepared sections are closely studied.

\17ithoat clescrihing the method whereby the nutritive ma- terials a rc carried into the derelopiug egg, Jorgensen devotes a considerable portion of his accoiiiit to a description of a precipitation membrane (~iederschlagsmemhraii) which is said to form between tlic original egg protoplasm (Original- plasma) and the nutritive material (Niihrplasma) supplied by tlie iiiirse cells. Here, again, it appears that faulty fixa- tion may he responsible foi* most of the phenomena involved. In figure 3, for example, ~vhicli closely resembles some of Jor- gensen’s figures, a definite membrane appears to be present in the interior of the cell, but under high magnification sucli

EGG FORMATION I N A LEECH 341

sheets are resolved into a finely granular mass which, step by step, can be followed into the characteristic reticulum of later stages.

LITEKATURE CITED

JORGENSEN, Max 1913 Zellenstudien. 11. Die Ei- und Kahrzellen von Piscicola. Areh. f . Zellforsehung., Bd. 10.

KORSCHELT UXD HEIDER 1902-1903 Lehrbuch der vergleichenden Entwicklungs- geschichtc, Allgemeiiier Theil. Fisclier, Jena.

WILSON, E. B. 1925 The cell in development and heredity, 3rd ed. (contains full bibliography and comprehensive review of the subject of ova with accessory cells).

PLATE 1

EXPLANATION OF FIGURES

1 Mass of oogonia forming ovum-nurse cell groups. f, follicle. ‘7

3 4 5

G

Half -developed ovum with superficial pseudopodial processes and network

Young ovum, showing cliaracteristic distribution of the nutritive material. Nurse cells in early stage of granule (mitochondrialg) formation. Supporting cell surrounded by nurse cells. Actual length of supporting cell

Nurse-cell group comprising central supporting cell and young ovum ( a t

of granules entering from the nurse cell group.

nucleus, 0.045 mm.

bottom of figure) surrounded by a follicle cell. Diameter, 0.26 mm.

342

EGG FORMATION I N A LEECH HAROLD HEATH

PLATE 1

343

PLATE 2

EXPLdNATION OF FIGURES

7 8 Young oogoiiinl group. f, follicle; s, supporting cell. !I

10

11

Xutrit ire material passing through the vitellirie membrane into the egg.

Al)iiorm:il oogonial group, showing two cells embedded in the large sap-

Ovum nearing maturity with the nurse cells entering the final stages of

Ovum, with maturation spindle, surrounded l i y remnants of nurse cells and

porting cell while otliws are attached to its free surface.

(legenern tion.

follicular membmne. Diametcr, 0.27 mni.

344

EGG FORJIBTIOK I N A LEECH HAROJLJ H E \ T H

P L A T E 2

345