studies on the ovarian follicle cells of drosophila by r. c ......king and koch—ovarian follicle...

297

Studies on the ovarian follicle cells of Drosophila

By R. C. KING and ELIZABETH A. KOCH

(From Northwestern University, Evanston, Illinois, U.S.A.)

With I I plates (figs, i, z, 5, 8 to 12, and 14 to 16)

SummaryStudies are described of the ultrastructure of the follicle cells which invest the oocyteof Drosophila melanogaster at the time of vitelline membrane formation. Of particularinterest are organelles made up of endoplasmic reticulum organized into a husk ofconcentric lamellae which surround lipidal droplets. These epithelial bodies are seenonly at the time the vitelline membrane is being formed, and it is assumed thereforethat the lipidal material of the epithelial body may be utilized somehow in the fabrica-tion of the vitelline membrane. Cytochemical studies have shown this membrane tocontain at least 5 classes of compounds; a protein, two lipids (which may be dis-tinguished by differences in their resistance to extraction by various solvents), and2 polysaccharides (1 neutral and 1 acidic). Studies were made of vitelline membraneformation in the ovaries of flies homozygous for either of 2 recessive, female-sterilegenes (tiny and female sterile). In the case of the ty mutation vitelline membrane materialis sometimes secreted between follicle and nurse cells, while in the mutant fes vitellinemembrane is observed in rare instances to be secreted between follicle cells and anadjacent layer of tumour cells. In the latter case the vitelline membrane shows alteredcytochemical properties. The fact that vitelline membrane can be secreted by folliclecells not adjacent to an oocyte demonstrates that it is the follicle cell rather than theoocyte that plays the major role in the secretion of the precursor material of the vitel-line membrane. Subsequently the follicle cells secrete the egg-shell, or chorion, whichis subdivided into a dense, compartmented, inner endochorion, and a pale, outer exo-chorion. A description is given of the ultrastructure of the follicle cells during thesecretion of the endochorion and the exochorion. The endochorion contains a protein,a polysaccharide, and a lipid, all of which may be distinguished cytochemically fromthe vitelline membrane compounds. The exochorion contains large amounts of acidicmucopolysaccharides. Specialized follicle cells form the micropylar apparatus andthe chorionic appendages. The formation of the chorion and chorionic appendages isdiscussed in the light of information gained from abnormalities of the chorions andchorionic appendages seen in ty and/ j 2-1 oocytes. Subsequent to the time the eggleaves the ovariole a layer of waterproofing wax is secreted between the vitelline mem-brane and the chorion.

IntroductionOOGENESIS has been studied in several species of flies belonging to the genusDrosophila, and in these species (melanogaster, willistoni, pseudo-obscura, gib-berosa, and virilis) the general sequence of events is the same (King andWolfsberg, 1957; Burnett and King, 1962). The oocyte arises within thegermarium as a member of a group of 16 daughter cells which are producedby 4 consecutive divisions of an oogonium. The 16-cell cyst (which sub-sequently is enveloped by mesodermal cells) pinches off the germarium and[Quart. J. micr. Sci., Vol. 104, pt. 3, pp. 297-320, 1963.]

2421.3 X

298 King and Koch—Ovarian follicle cells of Drosophila

so becomes the first egg chamber in the ovariole. The oocyte, the most pos-terior cell of the group, is nourished by the 15 more anterior cells, which aredesignated as nurse cells. Intercommunication of cytoplasm between allmembers of the 16-cell cyst is made possible by large pores in the wallsseparating adjacent cells (King and Devine, 1958). Similar pore systems havebeen described in the egg chambers of other insects (Giinthert, 1910; Dederer,1915; Verhein, 1921; Hirschler, 1942).

The process of oogenesis in adult D. melanogaster has been described, andthe development of the egg subdivided into a series of consecutive stagesending with stage 14, the mature, ovarian, primary oocyte (King, Rubinson,and Smith, 1956). The time required for the egg to pass through each develop-mental stage has been estimated, and the relative rates of growth of the variouscomponents of the developing chamber have been calculated (King, 1957).

At first all 16 daughter cells grow at identical rates. However, duringvitellogenesis (stages 8 to 11) the oocyte grows at a rate 10 times more rapidlythan previously and at the expense of the nurse cells which shrink and even-tually degenerate. During stages 12 to 14 no further increase in the volume ofthe oocyte takes place, but great changes occur in the yolk organelles (King,i960). Between stages 1 and 14 the oocyte increases in volume over 100,000times, and under optimum conditions it takes 3 days for the completion of thisprocess.

The division, migration, differentiation, and secretory behaviour of normalfollicle cells as a function of developmental stage have been described recentlyby King and Vanoucek (i960) from a study of Feulgen-stained whole mounts(table 1). An initial 10-h period exists during which mesodermal folliclecells congregate posteriorly in the germarium to form an envelope of about 80cuboidal cells about the 16-cell cyst, which subsequently pinches off from,the germarium. Another 6 to 8 follicle cells form the stalk which connects thechamber to the germarium and eventually to the next chamber to be pinchedoff (fig. 1). During the next 30 h as the cyst grows in volume the envelopingfollicle cells divide and so keep abreast of the increase in surface area. How-ever, during stage 6 a maximum follicle cell number of roughly 1,200 is reached,and mitoses cease. Thus, if all follicle cells undergo a similar number ofdivisions, it follows that 4 consecutive divisions would supply all the cellsneeded. By the end of stage 7 the number of follicle cells is no greater thanit was 20 h earlier at the beginning of stage 6. The cells have increased inarea, however, and are homogeneously distributed over the chamber surface.During stage 8 (which lasts 4 h) a posteriorward migration of follicle cellsappears to occur over the surface of the chamber, with the result that abovethe nurse cells the number of follicle cell nuclei per unit area is reduced and

FIG. 1 (plate). A phase-contrast light micrograph of a section showing 2 stalks connectingadjacent egg chambers of D. willistoni. The arrow points to a nucleus of the epithelial sheath.The developing oocytes lie in single file in the lumen of the epithelial sheath cylinder. Seefig. 4 for the submicroscopic morphology of this structure. OsO4, formalin, CdCla; epon;azure B (pH 4). n5, nurse nucleus; n4, follicle cell nucleus.

f

FIG. I

R. C. KING and E. A. KOCH

FIG. 2


King and Koch—Ovarian follicle cells of Drosophila 299

TABLE I

The origin, division, migration, differentiation, and function of the follicle cellsof D. melanogaster. See text for an accompanying account. S = stage

designationGermarial mesoderm

Follicle cells

Migration Ienvelope cyst (S i )

Cell division(8o-»-iooo) (S 2-5)

Growth (S 6, 7)

IMigration II

over cyst surface (S 8)

/ +Numerous migrant Few stationary

posterior cells anterior cells ^ ^ ^f \j \ ^ ^^" Migration III of border

Anterior migratory Stationary columnar Squamous cells cells through nursecells cells (10 \i high) (005 p. high) sur- chamber (S 9, 10)

\ surrounding rounding nurse \Migration IV oocyte cell chamber Vitelline membrane

(centripetal) | (S 10, 11)(S 10, 11) Vitelline membrane \

I (S 9 to 11) Micropylar chorionChorionic appen- \ (S 12)

dages (S 12, 13) Inner endochorion(S12)

Outer endochorion(S13)

Exochorion (S 13,14)

the number above the oocyte increased. Eventually a 15-fold differencedevelops between the two regions with respect to the abundance of nuclei.As yolk formation commences those follicle cells above nurse cells becomeexceedingly thin (50 m/n is about the minimum thickness observed), while thoseabove the oocyte become columnar. By the end of stage 10 the columnarfollicle cells have reached their maximum heights. The heights observedrange between 10 and 30 /x depending upon the position of the cell, since (asone can see in fig. 2) the dorsal follicle cells become taller than the ventral

FIG. 2 (plate). A light photomicrograph of a sectioned late stage-10 egg chamber. Note thegreater height of the columnar follicle cells surrounding the dorsal portion of the oocyte. Theoocyte nucleus and border cells are also dorsal in position. An arrow points to flattened,basiphil cells from the anterior columnar epithelium which are migrating centripetally be-tween the oocyte and the adjacent nurse cell. OsO4) formalin, CdCl2; epon, azure B (pH 4).The left, upper, right, and lower axes of the micrograph mark the dorsal, anterior, ventral,and posterior axes.

King and Koch—Ovarian follicle cells of Drosophila

0 10

FIG. 3. A drawing of a sectioned egg chamber of D. melanogaster. The oocyte is in stage 10,but is not as far along in development as the chamber shown in fig. 2. «3, the nucleus of oneof the squamous follicle cells which compose the thin epithelium surrounding the nurse cellchamber. nls, a portion of the compound nucleolus of a nurse cell nucleus. Banded chromo-somes are also present within the nucleus. The border cells will migrate laterally until theycome to lie opposite the oocyte nucleus. They function later in the formation of the micro-pylar apparatus, p, one of the pores which allow communication of cytoplasm betweenthe 16 daughter cells of the cyst. Three other pores are shown, vit, vitelline membrane.This structure is laid down between the surfaces of the columnar follicle cells and theoocyte and between the oocyte and the border cells. It is not produced at the nurse cell /•oocyte interface. However, a centripetal migration of the most anterior, columnar follicle


ones. This difference in follicle cell height was brought to our attention byDr. Kulbir S. Gill of Yale University. The columnar cells function in theformation of the vitelline membrane (during stages 9 to 11) and subsequently(during stages 12 to 14) the chorion is laid down by the follicle cells upon theirsurface membranes. During stage 9 and early in stage 10 a group of 6 to 10follicle cells which originally resided at the anterior pole of the egg chambermigrates posteriorly through the central interior of the nurse chamber. Thesefollicle cells squeeze between the nurse cells and eventually reach the surfaceof the oocyte. They then migrate dorsally and come to lie opposite the oocytenucleus (contrast figs. 2, 3). The entire migration takes about an hour. These'border cells' first secrete vitelline membrane and later form the micropylarcomplex. During stages 10 and 11 anterior cells from the columnar layermigrate centripetally between the oocyte and adjacent nurse cells. This migra-tion takes about 20 min. The vitelline membrane is extended inwards betweenthe oocyte and nurse cells by these follicle cells. During stages 12 and 13some of these cells form the dorsal chorionic appendages. Maximum growthof follicle cells occurs during stages 9 to 12 (a time interval lasting aboutan hour). Subsequently as the chorion forms, the columnar follicle cellsdecrease in height.

In the remainder of this paper we will describe the microscopic and sub-microscopic anatomy of the follicle cells during the secretion of the vitellinemembrane and chorion, report some cytochemical properties of the vitellineand the basement membranes and the chorion, and describe some abnormal-ities in the location of vitelline membranes and in the morphology of thechorions and the chorionic appendages seen in the ovaries of flies homozygousfor certain recessive, female-sterile genes.

Material and methodsFemale D. melanogaster of various ages belonging to four genotypes

(Oregon-S wild type, Sfes++Alu Itj+fes dptx Sp+ + , bur fs 2-1, and^2 ty)provided the majority of the ovarian tissues studied. For descriptions of themutant stocks used see King, Koch, and Cassens (1961) and King and Burnett(1957). Inaddition, females from the Barbados-3, wild type strain of D. willis-toni provided material. The flies were reared at 25 ° C on the medium de-scribed by King and Wood (1955). The ovaries were dissected from etherizedflies immersed in Drosophila Ringer solution (the formula for this is given in

cells (which occurs later in stage 10; see fig. 2) will gradually extend the vitelline mem-brane inwards, k, the karyosome, a DNA-containing granule, characteristically found withinthe oocyte nucleus, ys, an alpha yolk sphere. n2, the nucleus of a columnar follicle cell.The cytoplasm of these cells contains large, sudanophil, epithelial bodies (here shown asopen circles) and smaller PA/Spositive droplets (here shown as solid dots) The sudano

that these enter the ooplasm and then disappear. The mitochondria of the nurse cells, folliclecells, and oocyte are not included in the drawing. The epithelial sheath of the ovariole is alsonot shown. The left, upper, right, and lower axes of the drawing mark the dorsal, anterior,ventral, and posterior axes.


Growth, 20, p. 121) and were transferred subsequently to a fixative. Amongthe fixatives used were OsO4 (Caulfield, 1957), KMnO4 (Luft, 1956), andKahle's fluid (Gurr, i960, p. 304). However, our best electron micrographscame from material fixed first in buffered 1% OsO4 for 10 min and then for10 min in buffered 10% formalin containing 1% CdCl2. These solutionswere made by preparing double-strength aqueous solutions of the before-mentioned ingredients and subsequently adding an aliquot of either solutionto an equal quantity of buffered salt solution. This solution consisted of2 parts solution A, 2 parts solution B, and 1 part solution C. Solution Awas a veronal acetate stock solution, solution B was N/10 HCI, and solution Cwas 3% aqueous MgCl2.6H2O. Methacrylate, vestopal W, and epon wereused as embedding media. The details of tissue processing for methacrylate-embedded material were generally the same as those described in King (1960).The procedure outlined by Tzitsikas, Rdzok, and Vatter (1961) was used forvestopal W, and that of Luft (1961) for epon. In the latter case the tissue wasinfiltrated with the resin mixture employing 2 parts epon-DDSA to 8 partsepon-NMA and polymerized according to Luft's slow schedule. Veronalacetate buffers, pH 7-4 (Gurr, i960, p. 274), were used, and fixations, de-hydrations, and infiltrations took place at 2° C.

Enzyme extractions were performed on z-fx sections cut from Kahle-fixedovaries embedded in methacrylate. Thick sections were cut using a Leitz-Fernandez-Moran microtome. The methacrylate was removed with xylene,and the sections were generally incubated at 370 C in various enzyme-containing baths (see table 2 for details). Controls were run with buffersolutions.

T A B L E 2

Data on the digestion procedures employed

Enzyme

trypsin

pepsin

papain

Source

bovinepancreas

hogstomach

papaya

Supplier

NBC

Sigma

NBC

pHused

7 3

2 0

7-o

Dissolved in

Michaelis B(Pearse, pg. 781)

001 N HCI

Sorensen B(Pearse, pg. 780)

Digestiontime

1, 24

i, 2

1

Aqueousconcentration

O'l, I'O

0 1

I ' O

KEY: NBC, Nutritional Biochemicals Corporation, 21010 Miles Avenue, Cleveland 28,Ohio; Sigma, Sigma Chemical Company, 3500 DeKalb Street, St. Louis 18, Missouri;Pearse—see references under Pearse (1960). In the case of papain KCN and EDTA (disodiumsalt of ethylene dinitrilotetra-acetic acid) were added to yield a final concentration of 5 mM,and the incubation temperature was 22° C. B = buffer solution. The buffer solutions wereused at full strength.

The aqueous staining solutions employed were azure B (0-025%or 9), fast green (o-i% at pH 2 or 8), Nile blue (1% at pH 2), and alcian blue

<\s

tiW^f

\, , w

i t

FIG. 4. A drawing of the ultrastructure of that region of the follicular epithelium shown by thearrow in figure 3. bvi, basement membrane, ifo, follicle cell / oocyte interface. Note theinterdigitating microvillar projections arising from the plasma membranes of both oocyte andfollicle cells and the developing vitelline bodies, iff, the interface between adjacent folliclecells. Note the stacks of tubular endoplasmic reticulum lying parallel to the plasma mem-branes of contiguous follicle cells. nit the nucleus of a follicle cell. Note the finger-likeinvagination of the nuclear envelope and the orderly cluster of dense particles below it. Wherethe nuclear envelope is cut tangentially annulate 'pores' can be seen. A dense central nucleolusis evident, g, Golgi material, eb, epithelial bodies surrounded by concentric layers of endo-plasmic reticulum. The dense material appears to be extracted from the cores of some.m, a mitochondrion in contact with a reticulate body, r, reticulate bodies surrounded by

R. C. K I N G and

whorls of endoplasmic reticulum. One reticulate body contains a dense droplet. ne, thenucleus of an epithelial sheath cell. Note the parallel clusters of smooth muscle fibrils inthe cytoplasm and the mitochondria and cross-sectioned trachcoles. t, a longitudinally sec-tioned trachea. Note that the corrugated border of the cell lining the tracheal lumen is coatedby a dense membrane. The dense, circular body found in each protruding fold is a sectionedtaenidium. At the upper portion of the figure the taenidia are cut tangentially. A sectionedtracheole devoid of taenidia is also present, o, the ooplasm contains pale, homogeneous alphayolk spheres which are enclosed by double-walled envelopes, mitochondria, stellate lipoidalbodies, Golgi material, lamellar stacks, and reticulate bodies. Alpha yolk spheres when cuttangentially show an orderly arrangement of particles on their surfaces.

E. A. KOCH


( 1 % at pH 2). Staining times for azure B and fast green were standardizedto \ h at room temperature (21 to 23° C) or heated to 6o° C. Excess dye wasrinsed from sections with a buffer solution of the same pH, and the excessbuffer was rinsed away with tap-water. The slides were then passed through3 changes of tertiary butyl alcohol and 2 changes of toluene before coveringthe sections with permount and a cover slip. The periodic acid / Schiff(PA/S) and Feulgen procedures were also used. The rationale and technicaldetails for the above cytochemical procedures have been discussed in a recentpaper (King, i960). Staining took place through the plastic in the case ofvestopal-embedded sections and generally in the case of methacrylate-embedded sections. Preceding enzyme extractions (or their controls) themethacrylate was, of course, removed, and the methacrylate was also removedwhenever alcian blue was used as a stain. This dye penetrates vestopal W butnot methacrylate or epon.

In some sections acidic groups were blocked by methylation. These sec-tions were left for 3 h at 6o° C in an acidified methanol mixture (0-3 ml con-centrated HCl+40 ml methanol). Phenylhydrazine was also employed in theblockade of carbonyl groups. Sections to be subjected to carbonyl blockadewere placed for 1 h in o*i N aqueous phenylhydrazine hydrochloride (pH 3)at room temperature. Aqueous o-i M thioglycollic acid (pH 11) was used tobathe sections in those attempts made at rupturing disulphide bridges linkingprotein chains. Thioglycollate treatments were performed either at roomtemperature (210 to 230 C) or at 37° C. The length of the treatment variedbetween 1 and 24 h at room temperature and between 1 and 10 h at37° C.

Electron micrographs were taken of thin sections (giving grey interferencepatterns) cut by an LKB ultrotome from the same plastic blocks whichpreviously provided thick sections for cytochemical studies, utilizing the lightmicroscope. Observations were made utilizing a Hitachi HS-6 (50 kV)electron microscope equipped with 10- or zo-fi platinum apertures. Themeasurements given in the body of the paper refer to those determined fromelectron micrographs. No attempt was made to correct for the shrinkagewhich accompanies cytological processing. Measurements made upon thelengths and widths of sectioned, mature, ovarian oocytes gave values 80 to85% of those taken from living, mature, ovarian oocytes.

Morphology of the follicle cells at the time of vitelline membraneformation

The drawing reproduced in fig. 3 shows the entire egg chamber as seenwith the light microscope at the time of vitelline membrane formation. Thedrawing reproduced in fig. 4 shows the morphology of a particular region ofthe follicular epithelium (shown by the arrow in fig. 2) as seen with theelectron microscope. The drawing is a composite made from 40 electronmicrographs, each of which showed a particular organelle or association ofsubcellular structures especially clearly.


Each columnar follicle cell is a regular, hexagonal prism about twice ashigh as it is wide. Its base is covered by a membrane about 0-3 p thick whichpresumably is secreted by the cell itself. The basement membranes ofadjacent follicle cells fuse together and so form a sheath which surroundsthe entire chamber and the interfollicular stalks connecting adjacent chambersas well. This sheath, which is sometimes called the tunica propria (Snod-grass, 1935), has the potential of serving as a selective dialytic membraneregulating chemical exchanges between cells of the ovariole and the haemo-lymph (a speculation first advanced by Bonhag and Arnold, 1961). The sur-face plasma membrane of the follicle cell is thrown into myriads of microvilliwhich interdigitate with similar microvilli produced by the plasma membraneof the oocyte. Within the intercellular space are formed numerous vitellinebodies, which eventually fuse to form the vitelline membrane, which servesas a flexible covering about the oocyte. No such microvilli are seen to originatefrom the adjacent plasma membranes of contiguous follicle cells. Subsequentto the fusion of the vitelline bodies droplets ranging in diameter between o-iand 0-3 jit are added to the vitelline membrane at the surface bordering thefollicle cells (fig. 5). These droplets form in areas filled with agranular, mem-branous vesicles of similar dimensions. The formation of the vitelline mem-brane takes roughly 1 h.

The nucleus of the follicle cell characteristically contains a few finger-likeinvaginations of its envelope which may extend inwards a micron or more.The nuclear envelope is studded with annular 'pores' each 40 to 50 m/n indiameter. The outer membrane of the nuclear envelope is coated with a singlelayer of tiny particles (presumably ribosomes). A single dense, amorphousnucleolus is present within the nucleus, and additional dense granules (largerthan the ribosomes) are seen which may represent sectioned chromosomalfilaments. According to Schultz (1956) nuclei from differentiated follicle cellscontain between 8 and 16 times the haploid amount of DNA. Thus replica-tion of DNA must continue after mitoses cease. Such replications presumablyaccount for the uptake of H3-thymidine by the nuclei of columnar folliclecells at stage 10 reported by Nigon and Nonnenmacher (1961).

The cytoplasm of columnar follicle cells contains small populations ofspherical or ellipsoidal, sudanophil bodies which are roughly 1 f/. in longestdimension. These epithelial bodies (King, i960) are found only in columnarfollicle cells and only at the time of vitelline membrane formation. Under theelectron microscope epithelial bodies are seen as circular profiles surroundedby an endoplasmic reticulum, which is organized into a husk of concentric

FIG. S (plate). An electron micrograph showing a late stage in the formation of the vitellinemembrane. The arrow points to one of many droplets which are fusing to the vitellinemembrane at its surface bordering the follicular epithelium. Note that microvilli are far morepronounced on the oocyte side of the vitelline membrane. A mature and two immature alphayolk spheres are seen in the ooplasm. The contents of the immature spheres appear to be lessresistant to extraction by the solvents the tissue encountered during dehydration and infiltra-tion than is that of the mature sphere. OsO4, formalin, CdCl2; epon; stained with leadhydroxide. The tiny specks represent an artifact and should be ignored.

FIG.



layers of double membranes, which contain particles on their outer surfaces.Varied configurations are assumed by the endoplasmic reticulum comprisingthese organelles. In some sections the outer layers of concentric lamellae areseen to be in close association with the plasma membrane or with the nuclearenvelope. These associations are not surprising in view of the considerablebody of evidence which suggests a fundamental functional, as well as structural,similarity between the plasma membrane and the intracellular membranes ofthe endoplasmic reticulum and nucleus (Meek and Moses, 1961). The epi-thelial bodies appear to arise by the accumulation of material betweenadjacent lamellae. This accumulation will sometimes give rise to goblet-shaped structures; while at other times material may accumulate in severalregions and between different layers in the concentric lamellar system to pro-duce more complex configurations. The material which accumulates intospherical or ellipsoidal droplets within the lamellar whorls is often dense andamorphous. Structureless, spherical, pale, central areas are often seen in thesedroplets, and these presumably represent areas where lipidal material hasbeen extracted. Perhaps the osmium failed to penetrate to the centre of alldroplets, and only lipid molecules cross-linked through osmium atoms arerendered insoluble to the solvents used during dehydration and embedding.Some lamellar systems surround paler bodies which contain a network ofvesicles, dense granules, and wisps of dense amorphous material. Sometimesdeposits of both types are found in the same lamellar system, and occasionallya smaller, amorphous, dense droplet will be seen at the centre of a reticulatedeposit (fig. 4). Such observations suggest that the reticulate deposits mayarise first and subsequently transform into lipidal deposits by conversion oftheir contents into osmiophilic material.

The organized swarms of agranular, membranous vesicles representing theGolgi apparatus are also observed in the cytoplasm of follicle cells. Theentire organelle is generally about o-6 fj. in longest dimension. It is sometimesseen nestled alongside the lamellar system of an epithelial body. The Golgisystem is thought to concentrate into secretion granules the proteins newlysynthesized by the ribosomes of the endoplasmic reticulum (Caro, 1961).

Filamentous mitochondria reside in large numbers in the cytoplasm. Pale,reticulate bodies unaccompanied by lamellar whorls are present in smallernumbers, and these may represent the PA/S-positive granules noted underthe light microscope in the cytoplasm of columnar follicle cells (fig. 3).Occasionally a mitochondrion is seen in contact with a reticulate body.

The background cytoplasm contains endoplasmic reticulum composed oftiny vesicles (each about 50 m/j. in diameter) and of tubules which often lie instacks parallel to the plasma membranes of contiguous follicle cells. Myriadsof free granules (presumably ribosomes) are present in suspension.

In fig. 3 some characteristic ooplasmic organelles are shown as well asa portion of the ovariole wall and an adjacent tracheal cell. More detailedaccounts of these structures are available elsewhere (King, i960, 1962;Locke, 1958).


Cytochemical properties of the vitelline and basementmembranes

The vitelline membrane contains proteins, lipids, and polysaccharides. InKahle-fixed, methacrylate-embedded material a protein (P6) is retained whichis extracted from osmium-fixed material. Protein P6 stains with fast green atpH 2. It resists digestion by endopolypeptidases such as trypsin, pepsin, orpapain. However, protein P6 is not retained after a 90-min treatment withthioglycollic acid at 370 C, followed by a 1 h digestion in 1% trypsin (a pro-cedure known to digest keratin). These results suggest that thioglycollic acidruptures the disulphide bridges linking adjacent protein chains, thus per-mitting the protein to unfold. The protein then becomes susceptible todigestion by trypsin. After a thioglycollic acid treatment of 10 h at 370 Cwithout a subsequent trypsin digestion, the vitelline membrane stains lessintensely with fast green, suggesting that alkaline hydrolysis can remove someP6. When thioglycollic acid is used at room temperature, treatments lastingas long as 25 h do not remove P6. If Kahle-fixed, methacrylate-embeddedsections are placed in buffered aqueous 1% KMnO4 for 15 min, the proteinof the vitelline membrane binds Mn and subsequently fails to stain with fastgreen. The methylation procedure removes much Mn and at the same timerestores the fast green stainability of the vitelline membrane.

Two polysaccharides have been identified in the vitelline membrane. Thefirst is PA/S positive, alcian-blue negative, and resistant to TCA extraction.It is present in osmium-fixed, methacrylate-, or vestopal-embedded sectionsof oocytes. This neutral polysaccharide, previously designated by King{i960) as C5, is presumably responsible for the binding of lead by the vitellinemembrane (fig. 17, ibid.). The second acidic polysaccharide, hereafter calledC7, is alcian-blue positive and is retained in KMnO4-fixed material. Methyla-tion greatly reduces the alcian-blue stainability of C7. The vitelline membranebinds considerable Mn, if fixed with KMnO4. This binding is presumablythe result of the interaction of P6 and C7 with Mn. The KMnO4-fixed vitel-line membrane fails to bind fast green at pH2 or azure B at pH 9, indicatingthat Mn has masked most charged groups.

Lipids belonging to at least two classes reside in the vitelline membrane.An acidic lipid is found which has been designated by King (i960) as L3.It stains instantaneously with Nile blue at room temperature and is retainedin the mature vitelline membrane after all the fixation and embedding pro-cedures employed. In osmium-fixed, methacrylate-embedded sections theacidic lipid of the mature vitelline membrane resists a i-h extraction withacetone, chloroform, ethanol, or toluene at 6o° C; whereas some lipid isremoved from developing membrane such as that seen in stage-10 oocytes.The vitelline membrane is leached out of brominated, osmium-fixed oocytes,and it is likely that under normal conditions it is an osmium-L3 complexwhich assures retention of this membrane when osmium serves as thefixative.


The second lipid (hereafter designated as L7) is retained in osmium-fixed,methacrylate-embedded ovaries, but is lost from osmium-fixed, vestopal-embedded sections. It is the loss of L7 from vestopal material which pre-sumably is responsible for the fact that the vitelline membrane appears palerin electron micrographs taken from vestopal- than from methacrylate-em-bedded ultra-thin sections. Since osmium interacts with L7, it presumablycontains ethylenic linkages. L7 stains with fast green at pH 2. Originally L7

was thought to be a protein and was designated as P3 (King, i960). Subse-quently it was found that a 3-h extraction in 6o° C methanol removed L7. Thelipid is also sensitive to treatment with SO2-water, and as a result fails to bindfast green following the PA/S or Feulgen procedure. It may be that L7

belongs to that class of compounds known as proteolipids. These compoundscontain protein and lipid and are soluble in organic solvents.

The vitelline membrane in KMnO4-fixed, vestopal sections stains red withSchiff's reagent. This staining is greatly reduced by phenylhydrazine treat-ment. However, if a PA/S procedure is performed on sections previouslyexposed to phenylhydrazine, the vitelline membrane again stains red. Theseresults suggest that KMnO4, acting as an oxidizing agent, has liberated car-bonyl groups from carbohydrates which react with Schiff's reagent. Thesecarbonyl groups are masked when reaction with phenylhydrazine producesphenylhydrozones. However, not all the 1,2-glycol linkages contained in thecarbohydrate are cleaved by KMnO4, and these subsequently are convertedto dialdehydes by periodic acid and are free to combine with the Schiffreagent.

The vitelline membrane is PA/S positive in osmium-fixed, vestopal- ormethacrylate-embedded sections and in KMnO4-vestopal sections. Kahle-fixed, methacrylate sections show PA/S negative vitelline membranes.Kahle's fluid contains formalin, which is known under certain conditions toblock the PA/S reaction. Pearse (i960, p. 245) has pointed out that alkalinehydrolysis often increases the colour developed in the PA/S reaction byreversal of formalin blockade. However, alkaline hydrolysis (aqueous 0-2 NNaOH for 15 min at 220 C) preceding the PA/S procedure fails to permitcoloration of Kahle-fixed vitelline membranes. It follows that C5 and C7 areabsent from Kahle-fixed, methacrylate-embedded material.

Table 3 summarizes the data on the various cytochemically distinguishableclasses of substances retained by the vitelline membrane following a varietyof fixation and embedding procedures.

The basement membrane contains a protein which, like P6, resists digestionby trypsin, pepsin, and papain. However, the protein appears to be morebasic than Pe, since it stains with fast green at pH 8 following esterification ofits negatively charged groups by the methylation procedure, whereas theprotein of the vitelline membrane remains uncoloured. Furthermore, thebasement membrane protein is retained in osmium-fixed material, unlike P6.The basement membrane contains 2 cytologically distinguishable carbo-hydrates. One of these is PA/S positive and alcian-blue negative. It differs


TABLE 3

Retention of various classes of chemicals in the vitelline membrane as a functionof the fixation and embedding procedure

Fixative

OsO4OsO4KahleKMnO4

Dehydrant

alcoholacetonealcoholacetone

Embeddingmedium

methacrylatevestopalmethacrylatevestopal

Pe

+

++

?

c7

+

11

1 ++++

+

KEY: — = extracted; + = retained.

from C5 in that it is retained in Kahle-fixed basement membranes. Thesecond carbohydrate is acidic and resembles C7. There is no evidence fora lipid in the basement membrane. The basement membrane of Kahle-fixedfollicle cells binds Mn and subsequently fails to stain with fast green. Treat-ment of the section with SO2-water produces no visible reduction in thebrown coloration due to the bound Mn. However, fast green stainability isrestored.

The background cytoplasm of the columnar follicle cells contains a poly-saccharide (referred to as C2 by King, i960) which binds alcian blue. C2 andC7 differ in that C2 is retained after Os04-fixation, whereas C7 is retained afterKMnO4-fixation. Perhaps C2 serves as a precursor to C7.

Vitelline membrane formation in mutant ovariesThe question naturally arises as to whether the follicle cell or oocyte plays

the more active role in the secretion of the vitelline membrane. No organellehas been observed in the oocyte, which is present only at the time of vitellinemembrane formation. In the columnar follicle cell, on the other hand, the epi-thelial bodies and their investing whorls of granular lamellae are seen onlyat the time vitelline membrane is being formed. This finding suggests thatthe lipidal material of the epithelial body may be utilized somehow in thefabrication of the vitelline membrane. However, the epithelial body containsneither L3 nor L7> and vitelline membrane (unlike the epithelial body) failsto colour intensely with Sudan black B. Since the epithelial and vitellinebodies differ in their cytochemical properties, the former may serve as a pre-cursor for the latter, but during this conversion the former must undergochemical modification. Recall also that tiny droplets are seen within thefollicle cells (fig. 5) at the time the vitelline bodies are fusing. These dropletscongregate at the surface of the follicle cells bordering the vitelline membraneand subsequently fuse to the latter.

Further evidence on the relative roles played by the follicle cell and oocyteduring the secretion of the vitelline membrane has been obtained from studiesof abnormal oogenesis in females homozygous for either of two recessive,


female-sterile genes. The mutants are tiny (ty, 1-44-5) a n d female sterile(fes, 2-5±).

The ovaries of females homozygous for ty show a marked retardation ofvitellogenesis(King and Burnett, 1957).Furthermore the posteriorward migra-tion of follicle cells during stage 8appears to be abnormal (King andVanoucek, i960). The result is thatthe nuclear density over the posteriortwo-thirds of the chamber is greaterthan that above the anterior third. Thefollicle cells in the area of high nucleardensity differentiate into columnarcells; whereas the rest remain cuboidalor become squamous. Consequentlythe layer of columnar follicle cells ex-tends abnormally far forward and en-velops about one-half of the nursechamber (fig. 6). Rare instances havebeen observed where vitelline mem-brane was secreted between thecolumnar follicle cells and the adjacentnurse cells. Such regions are markedby arrows in fig. 6.

Hereditary ovarian tumours occur infemales of D. melanogaster homoxygousfor the 2nd chromosomal, recessive genefes. The ovarioles of such females aresubdivided into a series of sausage-shaped masses of cells, and many of a nurse cell. The arrows point to vitellinethese cells are in various Stages of - ^ b r a n e materi.l secreted between the

6 follicle cells and the adjacent nurse cells,mitosis. Some metaphases are multi-polar and others show a high degree of polyploidy (King, Burnett, andStaley, 1957). In a few cases egg chambers have been observed where vitellinemembrane was secreted between the columnar follicle cells and a layer ofundifferentiated tumour cells (fig. 7). Similar bodies have also been observedin tumorous chambers in females of D. melanogaster homozygous for the firstchromosomal, recessive gene fused (P. Smith, unpublished). The vitellinebodies are smooth on the surface facing the tumour cells but are irregular incontour at the surface facing the follicle cells. This observation, perhaps, canbe interpreted to mean that vitelline membrane material is being suppliedby the follicle cells, and as it flows outward from them it assumes a smoothoutline on its leading edge. Initially normal vitelline bodies are irregular incontour both on the surface facing the follicle cells and the surface facing theoocyte. However, by stage ro the microvilli formed by the follicle cell plasma

FIG. 6. A drawing of an ovarian chamberfrom a 6-day-oldg2 ty female. n7l the oocytenucleus; nl2, the nucleolus of the nucleusof a columnar follicle cell; ys, alpha yolksphere; n/3> the nucleolus of the nucleus of

3io King and Koch—Ovarian follicle cells of Drosophila

FIG. 7. A drawing of ovarian chambers from a 15-day-old S fes+Alu Itj+fes dp1* Sp+-\-female. The smaller chambers show the more typical situation where the follicle is filledwith hundreds of oogonia-like cells and surrounded by a layer of cuboidal cells. The folliclelayer is abnormal in that it is multilayered in some regions. The follicular epithelia of thelarger chambers contain in several regions columnar follicle cells 5 to 10 ft thick. In theintercellular space between these and the outermost tumour cell layer vitelline bodies haveformed, bm, the basement membrane of a follicle cell; tu, tumour cell; eb, epithelial body,c, nurse-like cell (another cell of similar morphology is seen in the central chamber); vit,vitelline body; d, degenerating cellular debris at the posterior pole of the chamber.

membrane are no longer conspicuous, and the surface of the vitelline mem-brane facing the follicle cells is now relatively smooth. At this stage the plasmamembrane of the oocyte still bears many microvilli on the surface facing thevitelline membrane (fig. 5). Thus the microvilli formed by the plasma mem-

Fm

R. C. KING an

A. KOCH


brane of the oocyte disappear somewhat later than do those of the follicle cells.By stage 12, however, the oocyte microvilli have been replaced by long foldsin the plasma membrane which project into the interior of the oocyte, andthus point in a direction opposite to that in which the microvilli pointedpreviously (King, i960, his fig. 17).

The above observations lead us to suggest that the columnar follicle cellafter reaching a certain stage of maturity manufactures much of the precursormaterial utilized in the formation of the vitelline membrane. This materialfinds its way to the intercellular space bordering the surface of the cell.Normally the plasma membrane of the oocyte forms the opposite border ofthe intercellular space; but under abnormal circumstances a plasma membranesupplied by some other cell may serve this purpose. We thus tend to disagreewith Okadaand Waddington (1959), who maintain that the vitelline membraneis secreted by the oocyte.

Cytochemical examination of the vitelline membrane produced in theovaries of fes jfes females reveals differences from the wild type membrane.The normal vitelline membrane fails to stain with fast green provided thetissue was fixed in OsO4 and embedded in vestopal (table 3). However,the vitelline membrane secreted by fes follicle cells and processed as describedabove does stain. The membrane binds fast green whether secreted betweenthe follicle cells and tumour cells or between follicle cells and an oocyte.

Normally only the follicle cells adjacent to the oocyte (namely, the bordercells and the follicle cells destined to become columnar) differentiate into cellscapable of forming vitelline membrane precursor material. This fact suggeststhat the oocyte normally induces this type of differentiation in follicle cells.The nature of this morphogenetic stimulus and those which in certainmutants cause similar types of differentiation in follicle cells not adjacent tooocytes remain to be explored.

The fact that follicle cell envelopes are found surrounding aggregations oftumour cells demonstrates that follicle cells will envelop groups of such cellseven though they lack the ability to differentiate either into nurse cells oroocytes. Thus a multicellular cyst does not have to contain differentiatednurse cells and an oocyte in order for follicle cells to surround it.

Morphology of the follicle cells at the time of chorion formationThe chorion, which forms a hard, dense protective outer shell about the

mature oocyte, is seen in electron micrographs to consist of an electron-dense,compartmented endochorion which faces the vitelline membrane, and a pale,outer exochorion. The endochorion is made up of a thin, inner layer (which

FIG. 8 (plate). A composite electron micrograph showing the mature chorion of D. willi-stoni. p, plasma membrane of follicle cell; endt, outer endochorion; endit inner endochorion;ends, endochorionic space; ex, exochorion; m, clustered mitochondria; n4, the nucleus; me,convoluted membranous system; t, tracheole cell; c, epithelial sheath cell. OsO4, formalin,CdCl6; epon.


rests upon the inner surface of a membrane which we assume to be the originalplasma membrane of the follicle cell) and a thicker outer layer. Pillars andsepta connect the 2 layers and so form a series of compartments. The air-filled endochorionic spaces within the compartments serve a respiratory func-tion for the developing embryo (Hinton, 1960a).

In the composite electron micrograph reproduced as fig. 8 the inner endo-chorion and the plasma membrane can be seen to have pulled apart in severalregions. Above the exochorion a layer of basiphilic, cytoplasmic material ispresent which varies considerably in thickness. In some of the thicker areasprofiles of nuclei, clustered mitochondria, and convoluted double-layeredmembranous systems are evident.

Compartmented endochorion is first seen with the light microscope at theextreme anterior and posterior surfaces of the egg about the time the dorsalappendages begin to form. Numerous droplets arise in the cytoplasm of thefollicle cells in the regions adjacent to the vitelline membrane (fig. 9). Thesedroplets adhere to the plasma membrane and to one another and so producea foam-like layer. It is postulated that the compartmented endochorionresults from the solidification of a cementing substance laid down in the inter-stices between these tightly packed vesicles. Under the light microscope thehexagonal pattern left by each follicle cell can be seen on the endochorionsurrounding the oocyte and less clearly on that of the appendages. Note infig. 10 that each hexagon has a stippled interior and is surrounded by a clear rim.The stippling results from sectioned endochorionic septa and struts viewedend-on, while the clear border areas represent intercellular spaces which are,of course, free of endochorion, since endochorion is an intracellular product.

Fig. 11 is a composite electron micrograph showing representative folliclecells early in the period of exochorion synthesis. Epithelial bodies are nolonger present, and the endoplasmic reticulum is organized into a compactcluster of small vesicles and tubules within which are embedded numerousmitochondria. Ellipsoidal and spherical multivesicular bodies (resemblingthose described in 1959 by Sotelo and Porter) have made an appearance.Stellate, osmiophilic, lipid droplets are sometimes seen either alone or inclose association with mitochondria. Both the lipid droplets and the multi-vesicular bodies are about 0-5 JU. in longest dimension. The arrow in fig. 11points to a cluster of heterogeneous organelles surrounded by a thin mem-brane. This cytoplasmic inclusion has overall dimensions of 1-5x2 /x. The

FIG. 9 (plate). A and B, light photomicrographs of the follicular epithelium at the time ofthe secretion of the endochorion. The follicular epithelium is to the left, the denser ooplasmto the right. Note in A the array of droplets in the follicle cell cytoplasm adjacent to theoocyte. B shows a later stage in this process. The interface between the follicle cells andoocyte is cut more tangentially in this section. A and B are at the same magnification. OsO4,formalin, CdCl2; epon; PA/S, Azure B (pH 4). c, an electron micrograph of the follicle cellcytoplasm at a stage similar to those shown above. Note the numerous membrane-encloseddroplets which correspond to the droplets seen in the light micrographs. The denser bodiesare mitochondria. OsO4, formalin, CdCl2; epon.

FIG. 9


FIG. IO



packet contains numerous smaller bodies, and the profiles of 15 of these areseen in the section. Serial sections through the packet show within it 50 or sosmaller ellipsoidal bodies of rather diverse morphology and a larger lipoidaldroplet as well. The packet does not appear to be attached to the nuclearenvelope. The ellipsoidal bodies within the packet are themselves comprisedof varying numbers of smaller vesicles. It is conceivable that the entire struc-ture represents a bizarre differentiation of the Golgi material. Packets ofsimilar morphology have been described in the mucus-secreting cells of thegill epithelium of the Axolotl by Schulz and de Paola (1958) (see their fig. 11).During the formation of the exochorion (fig. 12) the cytoplasm containsmyriads of agranular vesicles which apparently secrete and become engulfedby the polysaccharide which forms this outer coating. It is during this timethat beta yolk spheres appear in the ooplasm (fig. 13).

It is perhaps worth stressing the extensive development undergone by themembranous systems of follicle cells during oogenesis in contrast to thenurse cells and immature oocyte where no complex lamellar systems develop.However, it is not surprising that such is the case, considering the continuoustransfer of material which takes place from the synthetically active, endo-polyploid nurse cells to the oocyte. Complex systems of cytoplasmic lamellaewould certainly impede such a transfer of nutrients by blocking the poresystem (fig. 3) and thus would have a negative selective value.

Special differentiations of the chorionThe chorion is modified at the anterior pole of the oocyte to provide for

the fertilization of the egg (fig. 13). The endochorion forms a non-compart-mented protuberance, the chorionic micropylar cone, which contains a tubularcanal, the micropyle. The ooplasm and its surrounding vitelline and plasmamembranes protrude into the micropylar cone to form a nipple-like projection(King, i960). The endochorion of the micropylar cone is surrounded by anouter layer of exochorion. The border cells (table 1) secrete both the vitellinemembrane and chorionic portions of the micropylar apparatus. Cells similarto the border cells form the micropylar apparatus of other dipterans (Kor-schelt, 1889; Gross, 1903; Pantel, 1913; Nicholson, 1921). The chorion canbe dissolved by treatment with sodium hypochlorite. In such dechorionatedeggs a nipple-shaped protuberance surrounded by vitelline membrane marksthe former position of the chorionic micropylar cone.

The dorsal chorionic appendages (fig. 14, A) are another specialization ofthe anterior chorion. Each appendage consists of a cylinder of endochorionwhich encloses a strutwork of endochorionic pillars and septa and is enclosedby a thin layer of exochorion. These appendages serve as twin breathingtubes on occasions when the remainder of the embryo is submerged, and whenthe egg is completely covered by water the appendages extract oxygen fromit (Hinton, 19606). The chorionic appendages develop to each side of the

FIG. 10 (plate). A surface view of the endochorion of a mature Drosophila oocyte.2421.3 Y


degenerating nurse cells during stages 12 and 13. Between 90 and 120 folliclecells are responsible for the secretion of each appendage, and these cells areamong those which migrate centripetally between the oocyte and nurse chamberduring stages 10 and 11 (table 1). King and Vanoucek (i960) noted that a de-crease in the number of follicle cells covering the egg chamber occurred asthe oocyte matured. It is likely that the centripetally migrating follicle cellswhich were ignored in their calculations account for much of the 'loss'. Itappears that some of these follicle cells omit vitelline membrane productionand proceed directly with the secretion of endochorion and later exochorion.Perhaps the fact that they do not lie against the oocyte plasma membrane isresponsible for this omission of a part of their secretory repertory.

At the opposite pole of the egg the chorion forms yet another specializedarea (fig. 14, B) of unknown significance. In this posterior protuberance thecompartments within the endochorion are characteristically heightened.

Sufficient data are not as yet available to allow an accurate estimate of thelength of time required for the secretion of the chorion and its derivativesunder optimal conditions. However, it is clear that the endochorion is secretedvery rapidly (presumably in 10 min or less), whereas the exochorion is formedfar more slowly.

Cytochemistry of the chorionThe chorion and vitelline membrane show very clear-cut chemical differ-

ences. For example, a 20-min immersion of mature eggs in an aqueous 3%sodium hypochlorite solution will dissolve the chorion without affecting thevitelline membrane. Conversely, if ovaries are brominated prior to osmiumfixation, the vitelline membrane will be lost and the chorion retained inmethacrylate-embedded material (King, i960).

The endochorion contains a protein, a polysaccharide, and a lipid, hithertoreferred to as P2, C6, and L3, respectively (King, i960). The endochorionicprotein differs from that of the vitelline membrane in that it is retained inosmium-fixed material (table 3). Furthermore, while the treatment with thio-glycollic acid followed by trypsin removes the protein from the vitelline mem-brane, it does not remove the protein of the endochorion. The endochorionicpolysaccharide is alcian-blue negative and differs from the alcian-blue negativepolysaccharide of the vitelline membrane in that it is retained in Kahle-fixedmaterial (table 3). The endochorionic lipid (L2) and the vitelline membranelipid, which it resembles most closely (L3), differ in their solubilities in variousorganic solvents.

The exochorion contains large amounts of an acidic, alcian-blue-positive

FIG. 11 (plate). A composite electron micrograph showing follicle cells preparing for thesecretion of exochorion about a dorsal appendage. nt, follicle cell nucleus; n/j, follicle cellnucleolus; m, mitochondrion; mb, multivesicular body. Note that the cytoplasm is filled withvesicular and tubular elements of endoplasmic reticulum. The organelle indicated by thearrow may represent Golgi material (see text). The appendage lies above and the basal por-tions of the cells below the area illustrated. OsO4; vestopal.

FIG. II


end,

F I G . 12



polysaccharide. This material is retained in KMnO4-vestopal sections, but ismissing in Kahle-methacrylate sections, and its concentration is reduced inosmium-fixed, methacrylate-, or vestopal-embedded material. Certain dyessuch as alcian blue fail to penetrate epon. The sodium methoxide method ofMayor, Hampton, and Rosario (1961) successfully removes this resin fromsections, and alcian-blue staining of the exochorion can then be demonstrated.However, the sodium methoxide procedure removes the PAS-positivematerials from the vitelline membrane and causes dissociation of certain lipo-protein and glycoprotein complexes. Such dissociations result in enhancedstaining with fast green, Nile blue, and Schiff's reagent (following periodicacid oxidation). The methylation procedure when performed upon KMnO4-vestopal material abolishes alcian-blue stainability of all those structures whichnormally stain with this dye, except the exochorion. It follows that theexochorion must be extremely acidic.

Chorion of mutant ovariesIt is usual to find in the oocytes of homozygous tiny females that vitello-

genesis is greatly retarded. However, oocytes are produced in rare instanceswhich contain some yolk and are covered by a vitelline membrane andchroion. Such chorions are abnormal in that the endochorion is convolutedand deformed and is covered by an abnormally thick exochorion. The endo-chorionic peninsulas (shown in fig. 15) presumably arise in regions whereinfoldings occur in the plasmalemma of follicle cells before the secretion ofendochorion, and cytoplasmic droplets subsequently adhere to folded as wellas to normal membranes. Endochorionic projections perpendicular to thenormal chorion would result once such a system is covered with cementingsubstance. That the plasmalemma of the follicle cells is wrinkled in the firstplace suggests that a relatively fixed number of cells come to be above theoocyte as a result of migration (table 1), and these cells each synthesize a fixedamount of surface membrane irrespective of the volume of yolk beneath them.Chorions at earlier stages of development must be studied to see if the abovespeculations have any merit.

We conclude, therefore, that the processes of vitellogenesis and chorionformation are independent, at least to a certain extent. That is, vitellogenesismay be greatly retarded, and yet both the chorionic covering of the oocyteand the chorionic appendages are produced. However, for the secretion ofperfectly formed chorion and chorionic derivatives a normal volume ofooplasm seems to be required.

Females homozygous for fs 2-1 produce dorsal appendages which are some-times convoluted or fused (fig. 16). Such deformed appendages may resultfrom abnormal migratory routes being taken by the follicle cells responsible

FIG. 12 (plate). An electron micrograph showing a follicle cell at the time of exochorionformation. endu endochorion; ex, exochorion; t, tracheole cell; m, mitochondrion. Notethat the cytoplasm contains myriads of smooth-surfaced vesicles. At the exochorionic /cytoplasmic interface parallel stacks of lamellae are commonly seen. OsO4; vestopal.


for the formation of the appendages. In two localized anterior regions theendochorion under normal conditions is gradually extended forward to formthe dorsal appendages which lie to either side of the degenerating nurse cells.

10/A

FIG. 13. A drawing of the micropylar apparatus and associated structuresof the oocyte of D. melanogaster. app r, sectioned right chorionicappendage; mic, micropyle; end%, non-compartmented endochorion; ex,exochorion; app I, left chorionic appendage; n4, follicle cell nucleus; vit,vitelline membrane; bys, beta yolk sphere; end3, compartmented endo-chorion ; ays, alpha yolk sphere. The left, upper, right, and lower axesof the drawing mark the ventral, anterior, dorsal, and posterior axes. It isprobable that the micropyle also penetrates the vitelline membrane.

The follicle cells which perform this task appear to migrate between the nursecells and the squamous epithelium surrounding them. Follicle cells arrangethemselves in a circular cluster at the position destined to mark the point ofattachment of the dorsal appendage to the chorion. Each cell of the clustersecretes intracellularly in a centripetal fashion clusters of droplets whichcoalesce to form endochorion. As a consequence a tubular projection ofendochorion is produced surrounded by a follicular envelope. In an orderlyfashion other migratory follicle cells take their places distal to the earliermigrants and continue the secretion of endochorion. In this way the append-age is lengthened. The factors controlling the orderly migration and subse-

FlG. 14 (plate). A, a light micrograph of a developing chorionic appendage of a stage-13oocyte. app I, left appendage; app r, right appendage; 0, ooplasm; nb, nurse nucleus; nltfollicle cell nucleus. OsO4; vestopal; azure B (pH 4).

B, a phase-contrast photomicrograph of the posterior chorionic protuberance (marked byan arrow) of a mature ovarian, primary oocyte.

50/, - J

FIG. 14


FIG. IS



quent secretory behaviour of each follicle cell are enigmatic. However, theabnormal morphology of the appendages oifs 2 • 1 alluded to above demonstratesa genetic component in theprocess. Retardation of the migration of the folliclecells should result in short, blunt filaments. Such appendages are commonlyseen on oocytes produced by various other mutants of D. melanogaster(raspberry4, King and Burnett, 1957; singed36", Bender, i960; adipose, Doane,i960; and vestigial, David, 1961). A mutant characterized by rudimentaryappendages has also been described in D. funebris by Crew and Auerbach(1937). Blunt chorionic appendages appear on eggs laid by aminopterin-poisoned D. melanogaster (King and Sang, 1959).

Recently Throckmorton (1962) has summarized a study of the phylogenyof the egg filaments in the genus Drosophila (see his fig. 46). D. melanogasterand the other species belonging to the subgenus Sophophora produce eggsbearing two appendages. It is interesting to note that among this subgenusspecies are found where short, blunt appendages represent the normal situa-tion. Other species have long tapering filaments which remind one of thefilaments seen on the egg of D. melanogaster before it leaves the ovariole (sub-sequently the appendages take on a flattened oar-like shape). Among thespecies belonging to the subgenus Drosophila the common filament numberis 4. There are exceptions, however (3 filaments for species in the quinariagroup and one for the eggs of D. bandeirantorum). Species producing 4-fila-ment eggs belong to either of 2 groups; (a) those where the anterior pair offilaments are thin and fine while the posterior pair are thick and flattenedbasally, and (b) those where all 4 filaments are thin. The egg filaments ofspecies from the subgenus Pholadoris are irregular in position and variable innumber. It seems likely therefore that in the genus Drosophila varying geno-types exist which control the number and position of the migratory routestaken by filament-forming follicle cells, the size of the population of migratorycells, and the secretory activity of the cells. Furthermore the secretory activityof cells in the same region of different appendages sometimes proceeds atdifferent rates. In most species the genotype produces an invariable filamentpattern under the majority of environmental conditions. In a small numberof species, however, the genetic system is not so well buffered and variationsin the pattern commonly occur.

Envelopes of developing embryosIf stage 14 ovarian oocytes are placed in a saturated saline solution at room

temperature, they immediately shrink. However, the uterine eggs or laid eggsproduced by mated females do not shrink under these conditions, but theydo if saline at 450 C is used. Eggs laid by unmated females behave in the same

FIG. 15 (plate). A photomicrograph of an azure B-stained section of three adjacent oocytesfrom a 9-day-old female homozygous forg2 ty. Note the thickened, highly convoluted chorionsurrounding densely staining ooplasm. Arrows point to abnormal endochorionic peninsulaswhich project into the paler exochorion.


way as do fertilized eggs. It appears therefore that upon leaving the ovariolethe permeability properties of the egg undergo a drastic change.

According to Davies (1948) the waterproofing of the egg of Lucilia sericatainvolves the secretion of a wax which has a critical temperature in the regionof 3 8° C and is situated between the chorion and the vitelline membrane.Beament (1946) reports that the egg of Rhodnius prolixus becomes waterproofupon the secretion of a layer of wax less than 0-5 /x thick between the chorionand the vitelline membrane. The critical temperature for this wax is 42-5° C.In Aedes aegypti a critical temperature of 56° C is found for a wax which servesthe same purpose (Christophers, 1960). Salkeld and Potter (1953) report thatthe vitelline membrane of the egg of Diataraxia oleracea is held to the chorionby a waxy layer, and Mahowald (1962) has observed in early Drosophilamelanogaster embryos a layer of ether-extractable wax 50 m/LA thick lyingbetween the vitelline membrane and chorion. We have not seen such a waxlayer in ovarian eggs.

From the above information we conclude that regardless of whether or notfertilization occurs, a layer of waterproofing wax is laid down between thevitelline membrane and the chorion once the eggs leave the ovariole. Thewax undergoes a transformation in physical state when placed in a 450 Cenvironment, and as a consequence the egg becomes susceptible to dessication.Since the follicle cells have degenerated by the time the waxy layer is formed,they can play no role in its formation. If the wax is secreted by the oocyte itis difficult to see how it passes through the vitelline membrane. It is assumedthat the accessory glands play no role in the formation of the waxy layer, sincethe eggs produced by lozenge3ilc females resist shrinkage when placed in salinesolutions, and lozenge34k females lack accessory glands.

Moscona (1950) has demonstrated for the stick insect, Bacillus libanicus,that minerals are transferred from the chorion to the embryo during embryo-genesis. Wilson's analyses of Drosophila egg-shells (i960) reveal that they arerich in minerals, and a similar mineral transfer may take place during Droso-phila embryogenesis. However, no information on this subject is available atthe present time.

Our studies have demonstrated that the follicle cells which envelop thedeveloping oocyte of D. melanogaster are involved in the formation of a base-ment membrane, the vitelline membrane, the endochorion, and the exo-chorion, and that these membranes differ greatly from each other in theirchemistry and morphology. The follicle cell therefore offers excellent possi-bilities for studying the morphology of secretion in a complex submicroscopic

FIG. 16 (plate), A, a light micrograph of the abnormally convoluted dorsal appendages seenin a section from the ovary of a 5-day-old bur fs z-i female. OsO4, formalin, CdCI2; epon;PA/S, azure B (pH 4). n6, nucleus of degenerating nurse cell.

B, a light micrograph of 'Siamese twin' appendages seen in a section from a 6-day-oldbur fs Z"i female. The appendages originate and end individually, but are fused along theirmiddle segments. OsO4; methacrylate, fast green.

FIG. 16



system which during development engages in a succession of different syn-thetic activities. We can add to these advantages the availability of mutantgenes which disturb the functioning of the follicle cells, the potentialities ofaffecting follicle cell behaviour by modifying the nutrition of females (Sangand King, 1961), and the possibility of making in vitro radiotracer studies(King and Falk, i960) of the biosynthesis of macromolecules. In the futurewe hope to take advantage of some of these latter potentialities.

This research was supported in part by the National Science Foundation(research grant NSF-G11710) and by the U.S. Public Health Service (grantRG-9694). A portion of the research was completed during tenure by E. A. K.of a predoctoral fellowship (GPM-18847) from the Division of GeneralMedical Science, U.S. Public Health Service. The photographs used asfigs. 1,5,8, and 9, c were made by Mr. Richard P. Mills during tenure of anundergraduate research participation programme award from the NationalScience Foundation. The authors are grateful to Mrs. Peter Pakeltis for herconscientious assistance and to Mr. E. John Pfiffner for drawing fig. 4. Themanuscript was read critically by Drs. E. H. Slifer, V. B. Wigglesworth,J. R. Baker, M. Locke, and A. P. Mahowald.

ReferencesBEAMENT, J. W. L., 1946. Proc. roy. Soc. B, 133, 407.BENDER, H. A., i960. Genetics, 45, 867.BONHAG, P. F., and ARNOLD, W. J., 1961. J. Morph., 108, 107.BURNETT, R. G., and KING, R. C , 1962. J. Insect Pathol., 4, 104.CARO, L. G., 1961. J. biophys. biochem. Cytol., 10, 37.CAULFIELD, J. B., 1957. Ibid., 3, 827.CHRISTOPHERS, R., i960. Aedes aegypti (£,.) The yellow fever mosquito: Its life history, bio-

nomics, and structure. Cambridge (University Press).CREW, F. A. E., and AUERBACH, C , 1937. Proc. roy. Soc. Edinb., 57, 255.DAVID, JEAN, 1961. Bull. Biol. Fr. Belg., 95, 34.DAVIES, L., 1948. J. exp. Biol., 25, 71.DEDERER, P. H., 1915. J. Morph., 26, 1.DOANE, W. W., i960. J. exp. Zool., 145, 1.GROSS, J., 1903. Zool. Jahrb. (Anat.), 18, 71.GONTHERT, T., 1910. Ibid., 30, 301.GURR, E., i960. Methods of analytical histology and histo-chemistry. Baltimore (Williams &

Wilkins).HINTON, H. E., 1960a. J. Insect Physiol., 4, 176.

19606. Philos. Trans., ser. B, 243, 45.HIRSCHLER, J., 1942. Biol. Zentralb., 62, 555.KING, R. C., 1957. Growth, 21, 95.

i960. Ibid., 24, 265.1962. Drosophila Information Service, 36, 83.and BURNETT, R. G., 1957. Growth, 21, 263.and STALEY, N. A., 1957. Ibid., 21, 239.and DEVINE, R. L., 1958. Ibid., 22, 299.and FALK, G. J., i960. J. biophys. biochem. Cytol., 8, 550.KOCH, E. A., and CASSENS, G. A., 1961. Growth, 24, 45.RUBINSON, A. C, and SMITH, R. F., 1956. Ibid., 20, 121.and SANG, J. H., 1939. Ibid., 23, 37.and VANOUCEK, E. G., i960. Ibid., 24, 333.


KING, R. C , and WOLFSBERG, M. F., 1957. Growth, 21, 281.and WOOD, E. M., 195s- Genetics, 40, 490.

KORSCHELT, E., 1889. 'Zur Bildung der Eihiillen', Nova Acta Leopoldina: Abkandhmgen derDeutschen Akademie der Naturforscher {Leopoldina) Zu HallejSaale: Neue Folge, 183.

LOCKE, M., 1958. Quart. J. micr. Sci., 99, 29.LUFT, J. H., 1956. J. biophys. biochem. Cytol., 2, 799.

1961. Ibid., 9, 406.MAHOWALD, A. P., 1962. Drosophila Information Service, 36, 130.MAYOR, H. D., HAMPTON, J. C, and ROSARIO, B., 1961. J. biophys. biochem. Cytol., 9, 909.MEEK, G. A., and MOSES, M. J., 1961. Ibid., 10, 121.MOSCONA, A., 1950. Quart. J. micr. Sci., 91, 195.NICHOLSON, A. J., 1921. Ibid., 65, 395.NIGON, V., and NONNENMACHER, J., 1961. Developmental Biol., 3, 210.OKADA, E., and WADDINGTON, C. H., 1959. J. Embryol. exp. Morph., 7, 583.PANTEL, J., 1913. Cellule, 29, 1.PEARSE, A. G. E., i960. Histochemistry, theoretical and applied, 2nd edition. London (J. & A.

Churchill).SALKELD, E. H., and POTTER, C, 1953. Bull. Entomol. Res., 44, 527.SANG, J. H., and KING, R. C, 1961. J. exp. Biol., 38, 793.SCHULTZ, H., and DE PAOLA, D., 1958. Z. Zellforsch., 49, 125.SCHULTZ, J., 1956. Cold Spring Harbour Symp. Quant. Biol., 2i, 307.SNODGRASS, R. E., 1935. Principles of insect morphology, p. 511. New York (McGraw-Hill).SOTELO, J. R., and PORTER, K. R., 1959. J. biophys. biochem. Cytol., 5, 327.THROCKMORTON, L. H., 1962. University of Texas Publ. 6205, 207.TZITSIKAS, H., RDZOK, E. J., and VATTER, A. E., 1961. Stain Tech., 36, 335.VERHEIN, A., 1921. Zool. Jahrb. (Anat.), 42, 149.WILSON, B. R., i960. Ann. Ent. Soc. America. 53, 732.

studies on the ovarian follicle cells of drosophila by r. c ......king and koch—ovarian follicle...

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