Functional transformations accompanying the initiation of a terminal growth phase in the cecropia moth oocyte

DEVELOPMENTAL

BIOLOGY

Functional

17, 512-535

(1968)

Transformations

Initiation

of a Terminal

in the Cecropia WILLIAM Biology Department,

Accompanying Growth

Moth

the

Phase

Oocytel

H. TELFER AND LUCY M. ANDERSON’

University

of Pennsyluania,

Accepted

October

Philuddphia,

Pennsylvania

19104

18, 1960

INTRODUCTION

The oocyte and associated cells of an insect ovarian follicle undergo a closely integrated set of transformations as they complete their respective roles in yolk deposition and enter the terminal phases of egg production. The nurse cells, which characterize the follicles of many insects, discharge their highly basophilic cytoplasm into the oocyte (Bier, 1963)) sever their cytoplasmic connectives, and finally become pycnotic (King and Aggarwal, 1965). Shortly after this, the cortical cytoplasm of the oocyte completes its pinocytotic function in the genesis of the protein yolk spheres (Telfer, 1965)) and then proceeds to form the periplasm which, in the centrolecithal insect egg, will become the site of embryo formation. The follicle cells initiate the deposition of a chorion surrounding the oocyte (King and Koch, 1963) and in so doing may secrete as much as a quarter of the dry weight of the egg. On the assumption that this complex of morphological and functional changes will prove to be based on fundamental transformations in the synthetic and organizational machinery of the cells involved, an analysis of the termination of yolk deposition and the ensuing events which culminate in ovulation has been initiated. Of particular concern in this report is a set of changes associated with the cessation of blood protein uptake in the follicles of the cecropia moth. The contrasting behavior of the follicles as they terminate this process and enter a previously unexplored terminal growth phase was found to provide some unusual insights into the final stages of egg formation. 1 Supported by grants from Phi Beta Psi Sorority and the National Foundation ( GB-4463). ’ Predoctoral fellow of the National Science Foundation. 512

Science

TERMINAL

OOCYTEZ

METHODS

AND

Trypan Blue Uptake a.s an Indicator Cecropiu Moth Follicles

GROWTH

513

RESULTS

of Blood Protein

Sequestering in

For the purpose of this study an important feature of the lepidopteran ovary is that it may contain simultaneously and in linear order a graded sequence of intermediate stages of egg formation (Fig. 1). Two days before eclosion in the cecropia moth, the most mature follicle in each of the eight ovarioles contains an oocyte which has achieved its maximum size and has been enveloped by a chorion. Anterior to this are thirty or more follicles in graded stages of chorion formation and yolk deposition, and an uncounted number of previtellogenic follicles. The developmental time lag between successive follicles maturing at this time is 4-5 hours, although the interval tends to lengthen as the animal approaches eclosion (Telfer and Rutberg, 1960). A convenient way to identify those follicles in the series that are actively taking up blood proteins was found to be provided by vital staining with trypan blue. This procedure was suggested by the observation of Ramamurty (1964) that the ovaries of the scorpion fly can remove trypan blue from the blood and deposit it in the cortical yolk spheres in a manner resembling vitellogenic blood protein uptake. Ramamurty’s observations were confirmed and extended in cecropia females that had been injected during the final week of their pupalmoth transformation with a 1% suspension of trypan blue buffered at pH 6.2. Trypan blue is a polyelectrolyte with a net negative charge at physiological pII’s, due to its content of two sulfonic acid groups per free amino group. While it has a molecular weight of 960, it forms colloidal aggregates of indeterminate size when suspended in either distilled water or physiological salt solutions. Commercially obtained (Harleco) trypan blue was partially purified by dialysis for 24 hours against distilled water, followed by air drying in dishes. It was dissolved in a solution containing 0.020 M KCI, lo-.* hl CaCI,, lo-* M MgCI,, and 0.25 hl Tris-maleate. The purification procedure and the pH and ionic composition of the solvent were arrived at empirically as those most consistent with the viability of follicles in short-term cultures such as those to he described below. The dosage was 0.05 ml per gram body weight injected into the anterior abdominal segment of the developing adult. Dissections were performed with the animal immersed in a solution containing 0.04 M KCl. 0.015

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M M&l, 0.004 M CaCL, and 0.11 M tris-maleate buffer. The follicles were sufficiently viable for 30 minutes in this solution so that subsequent exposure to 1% trypan blue in short-term culture did not lead to the intense cytoplasmic staining characteristic of dying cells (Parker, 1961) . In 24 cases in which the animal was dissected within 1-4 hours after dye injection, the ovaries showed a characteristic pattern of staining (Fig. 1). Trypan blue had been accumulated by every follicle less than 2 mm long and large enough to contain yolk, as evidenced by an opaque ooplasm. Follicles longer than 2 mm were very lightly stained at the most; there was generally in each ovariole one or at the most two transitional Z-mm follicles showing an intermediate level of staining. Termination of staining usually occurred P6 follicles posterior to the point at which the nurse cells had atrophied. Histological examination of Bouin’s fixed or freeze-substituted ( Feder and Sidman, 1958) follicles revealed that the dye was localized in the most peripheral row of yolk spheres, as Ramamurty had reported for the scorpion fly. In three moths dissected 20-24 hours after the injection, the dyed yolk spheres continued to form a discrete layer overlying the deeper unlabeled yolk spheres, but the blue layer was proportionately deeper than after 3 hours as a result of a longer period of yolk deposition in the presence of the dye. In some cases, such as that shown in Fig. 2, there was a tendency for the stained yolk spheres to be smaller than those that had been produced prior to dye injection. Since a superficial stratum of small yolk spheres is not normally produced by the cecropia oocyte, reduced size appears to have resulted from exposure to trypan blue. This is confirmed by the fact that all the small yolk spheres and no others contained blue dye. Nevertheless, trypan blue had no other detectable effect on the rate of follicle growth, or the general cytological appearance of the cells. Thus, while trypan blue at the dosages used was not without influence on yolk sphere size, its interference did not detract from its convenience and effectiveness as an indicator of protein yolk deposition. The precision of trypan blue uptake as an indicator of the stages that sequester blood proteins was tested by immunochemical measurement of the amounts of two blood-derived yolk proteins extractable from the follicles. For this purpose animals on days 20-21 of the pupal-moth transformation were dissected 2 hours after dye injection. In the example to be described in detail here, the 20 most posterior and

TERXIISAL

OOCYTE

CROU’TII

51.5

F1c:. 1. The chain of follicles dissected from one of the eight ovarioles of .L The nurse cells appear as cccropia 11~0th female on da)- 20 of adult development. a white cap at the anterior pole of each of the hmaller Follicles. 1)arkening of the intermetlinte follicles resulted from vital staining with trypan 1~1~. x 3.2. Fn.. 2. A 10-p section of a follicle dissected from a moth 24 hours after injection with trypan blue. The follicle was fixed in Bouin’s solution, the best fixative thus far fo~md for preserving trypan blue in yolk q1herc.a. In thiq case, the tl\~l >.olk sph(~rc~s are sul)stantiall!~ smaller than those protlucc~tl prior ttr the inject&. X218.

mature follicles in each of the eight ovarioles failed to stain with trypan blue. Of these, approximately 10 appeared white due to chorion deposition, while the remainder were the usual bright yellow color of unchorionated and chorionating follicles. Anterior to these, the progressively less mature follicles were deeply stained. In order to provide sufficient material for the extraction, corresponding follicles from 4 ovarioles were pooled. Each set of 4 follicles was placed in 0.5 ml of 0.15 &I: NaCl buffered at pH 7.2 with phosphate and containing 2% sucrose (Preer and Telfer, 1957). The folliclths and their extraction medium were then frozen and thawed, a procedure which has been shown to lyse the yolk spheres and thus to release their contained blood proteins ( Telfer, 19f31). M’h en the contents of the thawred fol-

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TELFER

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ANDERSON

licles had been suspended by being drawn into and out of a pipette, and the solids had sedimented, the yellow color, due primarily to carotenoids conjugated with a blood-derived protein deposited in the yolk spheres (Telfer, 1961, 1965), remained in the clear saline extract. The extracts were assayed for their relative content of the two most prominent antigenic components of the yolk by Oudin’s antiserum-agar test (Telfer and Williams, 1953; Telfer, 1954, 1960). Since

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.

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stained

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I

I I

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I 1

unstained

I I ! I 2

2

4

6

charion

I I I i 8

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12

follicle number FIG. 3. The relative concentration of the female protein in extra&s. of follicles, plotted as a function of follicle position in the ovariole. The arrow indicates the largest and most posterior follicle that had accumulated trypan blue during -2 hours’ exposure to the dye.

a standard volume of medium was employed, the relative concentrations yielded by these tests could be taken as a direct index of the amount of the protein extracted from each set of follicles. Relative extracted amount of the sex-limited female protein (Telfer, 1954, 1960) is plotted in Fig. 3 as a function of follicle position. The position of the largest stained follicles from the four ovarioles is indicated by the arrow, while successively more anterior and posterior follicles are identified in numerical order. The data indicate that the maximum amount of this protein was extractable from the largest staining follicles, since subsequent developmental stages had not accumulated detectable additional amounts. The

second

most mature

staining

follicles

also yielded

the definitive

TERhfISAL

OOCYTE

GHOWTH

.51-Y

amount of extractable protein, While this mav indicate that the largest stained follicle had retained its trypan blue binding capacity fog several hours after it had terminated blood protein uptake, a contributing factor must also have been the 2-hour time lag between dye injection and ovariole dissection. It is likely, for instance, that the largest stained follicle had at the time of dissection ceased both trypan lrlue and blood protein uptake, and that the second largest follicl:~ was alreadvi close to terminating these activities. Thus. it seems fair to conclude that trypan blue uptake was nearly if not exactlv correlated with female protein uptake in this ovariolr. Assays of a seco’nd blood-derived yolk protein, the carotenoid protein, in the same extracts as those analyzed in Fig. 3 yielded the same relationships, as did both female protein and carotenoid protein assa~;s of follicle extracts from two additional animals.

Finding that the cessation of trypan blue uptake corresponds closely to the cessation of blood protein uptake means that follicles undergoing this fundamental transition can be identified with a precision and convenience that have never before been possible. Advantage was taken of this opportunity to inquire into what precisely is entailed in the termination of blood protein uptake, and what cvcnts follow in the formation of the egg. Attention is first directed to a set of phenomena occurring in the oocyte between the cessation of blood protein uptake and the first appearance of the chorion. That the two events are not simultaneous was indicated by histological analysis of follicles that had failed to stain during short t:rm exposure to the dye. In on:: animal injected on day 19 of development and dissected 2 hours later, for instance, a series of 5 or 6 unstained follicles in each ovariolc. representing between 20 and 30 hours of developmental time (Trlfer and Rutberg, 1960) showed no signs of a chorion. A more reliable estimate was possible in animals dissected 24 hours after dye injection. In 2 of 3 animals used for this purpose, histological analysis indicated that the most mature stained follicle in every ovariole examined contained a thin chorion adhering to the inner surface of the follicle cells (Fig. 4 ), as well as a layer of peripheral yolk spheres containing the dye. Since chorionating follicles are not stained after 2 hours’ exposure to trypan blue, we presume that these follicles terminated dye uptake shortly after the injection. This interpretation

518

TELFER

AND

ANDERSON

was supported by the fact that the more anterior follicles, which showed no evidence of chorion deposition, contained deeper layers of stained yolk spheres, indicating that they had spent a longer period of time accumulating blood proteins after dye injection. In all 8 ovar-

FIG. 4. Fluorescence microscope view of a section of the largest stained follicle in an ovariole dissected from an animal 24 hours after trypan blue injection. The red fluorescence of trypan blue upon ultraviolet illumination has proved to be more readily photographed than its blue color when it is present in small amounts. Fluorescence is shown here in the peripheral yolk spheres. The follicle cells, which are at the top of the picture, adhere to a slightly fluorescent layer of chorion. The separation of the chorion from the oocyte occurred during histological processing; it is a characteristic behavior of this stage of egg formation. OOC, oocyte containing yoke spheres; the arrow indicates the chorion adjacent to the follicle cells. Carnoy’s fixation. X 218.

ioles of the 3rd animal, the largest staining follicle had no detectable chorion, whereas its more posterior unstained neighbor did. In these three animals, therefore, approximately 24 hours had elapsed between the termination of trypan blue uptake and the initiation of chorion deposition. Analysis of the developmental events occurring during this period was stimulated by the completely unanticipated observation that the largest staining follicles in animals dissected after short-term injection were substantially smaller (Fig. 1) and of different shape than those that had commenced chorion formation. Measurement of the follicles

‘I’ERMISAL

OOCYTE

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519

in two animals that had been dissected 2 hours after trypan blue injection indicated that the oocytes of the largest staining follicles were shaped as prolate ellipsoids with average lengths of close to 1.9 mm and transverse diameters of 1.4-1.5 mm. The lengths of the chorionating oocytes, by contrast, were 2.4-2.5 mm and their widths along the dorsoventral axis (as defined by the prospective position of the enbryo ) were 2.1-2.2 mm. The increase in size was preeminently two dimensional, for the right-left axis in all chorionating oocytes vxs only 1.6-1.8 mm. Calculations of the volumes of ellipsoids with these diameters indicated that a volume increase of greater .than 50% had occurred between the loss of stainabilitv , and the onset of chorion formation. Thus, a third of the volume as well as the slightly flattened shape of the mature egg are generated in this 24-hour terminal growth phase.

i4rttoradiog~a~hic Analysis of the Teminal Grou:th Phaw Two important sources of materials contributing to the mass of the oocyte earlier in its development arc the nurse cells and. as we have seen, the proteins of the blood. Since the nurse cells atrophy before the termination of trypan blue staining and blood proteins are no longer accumulated, a different explanation of the terminal growth phase is required. In order to test the possibility that protein yolk spheres continue to be generated from endogenously synthesized materials, follicles in the terminal growth phase were analyzed autoradiographically after exposure to tritiated histidine. Histidine was employed because autoradiographic studies of cecropia vitellogenesis (Melius. 1966) have indicated that it is an effective indicator of endogenously synthesized components of the protein yolk spheres. A 6-gm animal on day 19 of development was injected with 0.15 mC of I.-histidine-2, 5-“H (Nuclear-Chicago) simultaneously with 0.15 ml of 1X trypan blue. After dissection 2 hours later, the follicles from two ovarioles were fixed in Bouin’s solution while those from a third were freeze substituted. The two methods yielded identical results for the objectives of this experiment. Paraffin sections 5 p thick were overlayered with Kodak NTB-2 liquid emulsion, and autoradiographs dcvelopcd after 8 weeks. In all follicles that had not initiated chorion deposition, the oocytcs contained a peripheral 10-15 p deep stratum of label, with the silver grains tending to cluster over what appeared to be small protein volk

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TELFER

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ANDERSON

FIG. 5. Autoradiograph of a follicle dissected from a 19-day developing adult 2 hours after injection with tritiated histidine and trypan blue. The follicle had stained with trypan blue. The silver grains overlying the follicle cells on the left

TERMINAL

OOCYTE

CH<)WTII

521

spheres (Figs. 5 and 6). Cortical deposition such as that detected autoradiographically by Bier (1963) for the protein yolk spheres of blow flies is therefore a feature of both staining and nonstaining follicles in cecropia. As in Bier’s results, label was not detectable in the more deep lying yolk spheres which had been produced prior to the injection. Aside from the structures in the oocyte cortex, the follicular epithclium was the most heavily labeled component of the follicle. Despite the occurrence of peripheral labeling in both stained and unstained follicles, there was a striking difference in its appearance and intensity. Labeling was substantially heavier in the unstained follicles of the terminal growth phase, and the cortical structures underlying the clustered silver grains tended to have diameters of less than 5 p, while the labeled yolk spheres of stained follicles were often as large as 8 p, In order to characterize the cortical bodies in terminal growth phase follicles more fully, they were examined in sections after a variety of methods of fixation and found to be exceedingly refractile structures relative to comparably sized yolk spheres of vitellogenic follicles. In glutaraldehyde-fixed follicles embedded in Epon, and sectioned at 1 I,,, they often had a ring-shaped appearance (compare Figs. 7 and 8). The rrfractile bodies appear, therefore, to be a new category of cortical structures that are generated after the cessation of blood protein uptake. The importance of endogenous histidine incorporation, rather thau are out of focus. The in-focus silver grains overlie yolk spheres in the oocyte cortex. Freeze substitution. ~545. FC, follicle cells; the arrow indicates a labeled \ olk sphere. FIG. 6. Autoradiograph of a nonstaining follicle from the same ovariole as that shown in Fig. 5. Cortical oocyte label is substantially heavier than that seen in Fig. 5. FC, follicle cells; the arrow indicates cortical labeling in the oocyte. Freeze substitution, X 545. Fro 7. Phase microscope view of the oocyte cortex and adjoining follicular epithelinm of a follicle sequestering blood proteins. FC, follicle cell; IS, intercellular space; the arrow indicates an immature cortical yolk sphere. Glutaraldrlwtlr fixation. x 1325. FIG. 8. Phase microscope view of the oocyte cortex and follicle cells of a terminal growth phase follicle from the same ovariole as that shown in Fig. 7. FC, follicle cell; arrows indicate an interfollicular cell space on the left, and a rcfractile hody in the docyte cortex on the right. The vitelline membrane between oorvte and follicle cells has begun to thicken. Glutaraldeh\dr fixation. x 1325.

522

FIG. 9.

TELFER

Autoradiqgraph

AND ANDERSON

of a terminal

growth

phase

follicle

that

had

been

exposed to tritiated histidine for 2 hours in culture. The band of heavy label is in the cortex of the oocyte. Follicle cells are to the left. Freeze substitution. x545. FIGS. 10-12. Autoradiographs of follicles dissected from an animal 22 hours

TERMINAL

OOCYTE

C,ROlt-‘i-II

52.3

labeled blood protein uptake, in the genesis of the cortical refractile bodies was confirmed by incubating unstained follicles for 2 hours in a culture medium composed of 5 parts of cell-free blood from females in the last week of development, one part of tritiated histidine (1 mC/ ml), and 4 parts of 6% bovine y-globulin in dissecting solution, which served as a diluent for the blood. The three smallest nonstaining folk cles from each ovariole were placed in 0.25 ml of the culture medium in a depression slide; after being sealed shut with a cover glass and paraffin, the depression slide was placed on a shaker for 2 hours. .i\utoradiograms of these follicles, after Bouin’s fixation, revealed ;I heavy band of label over the cortical cytoplasm of the oocyte with silver grains clustered in a manner identical 13ith that seen in unstained follicles labeled in situ (Fig. 9). Since only, ovarian tissue was included in the culture, aside from the possibilitv of low level contamination with bIood cells, the cortical labeling observed must have been due exclusively to endogenous ovarian actiTitv. jVhile %hour labeling established that cortical deposition occurs in oocytes throughout the terminal growth phase. it gave no indication of the total contribution of this activity to the volume increase of the oocyte. For this purpose autoradiograms were prepared of follicles exposed to label throughout their entire terminal growth phase. A 4-gm female on day 19 of development was simultaneously injected with 0.1 mC of tritiated histidine and 0.1 ml of 1% trvpan blue and dissected 2% hours later. In the most posterior stained Eolliclc, the cortical layel of rrfractile bodies was heavily labeled (Fig. 12). The adjacent yolk spheres, which contained no detectable label, showed a level of trypan blue which was too low to be detectable, by bright field microscopy, but could be visualized by fluorescence microscopy. Since a thin layel of chorion had also been deposited, this follicle had gone through the entirtl terminal growth phase in the presence of tritiated histidine. Even in this follicle, the superficial stratum of heavily labeled refractile bodies measured no more than $0 p drc~p. after injection with tritiatcd histidine and trypan blue. All folliclrs are from the same ovariole. In Fig. 10, the follicle shown had not yet entered the terminal growth phase at the time of fixation. That shown in Fig. 11 is a terminal growth phase follicle. The most posterior, stained follicle, which is shown in Fig. 12, had initiated chorion deposition. As in Fig. 4, the space between the chorion and the surface of the oocyte is an artifact that invariably occurs in follirl~s at this stage during histological processing. Carnoy’s fixation. x 218.

524

TELFER

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ANDERSON

The contrast in labeling with more anterior follicles in the same ovariole was striking, Autoradiograms of follicles Iess than 2 mm long, and therefore presumed to have sequestered blood proteins throughout the Iabeling period, revealed label in a 70-p deep band of yolk spheres with diameters of about 10 ,p (Fig. 10). There was an exact correspondence between the distribution of label and of trypan blue, with al1 labeled yolk spheres, and none others, containing sufficient dye to appear bIue in lo- I~ sections of Bouin’s fixed follicles. The only conspicuous difference between the appearance of label in these follicles and in comparably staged follicles exposed to tritiated histidine for only 2 hours was the substantially greater thickness of the band of labeled yolk spheres. Since a similar pattern of labeling with tritiated histidine had been observed in follicles from animals that had not been injected with trypan blue (Melius, 1966), the dye did not appear to alter the normal course of events leading to histidine incorporation in follicles in the protein uptake phase of growth. In the larger and more posterior follicles the stratum of labeled yolk spheres was overlaid by a 20-25 p thick layer of more heavily labeled refractile bodies with diameters of 2-5 p ( Fig. 11). The underlying stratum of labeled yolk spheres was progressively thinner in successively more posterior follicles while, as in the most posterior stained follicle, the more intensively labeled bodies continued to form a superficial layer 20-30 ,Uthick. Labeling of the refractile bodies was accompanied by equally heavy labeling of a membrane at the outer surfaces of the oocyte (Figs. 11 and 12). Since this structure ultimately lies between the chorion and the oocyte, it appears to be a vitelline membrane. Although a 0.1 p membrane has been seen at the same site in electron micrographs of follicles that were sequestering blood proteins (King and Aggarwal, 1965; Stay, 1965)) labeling at this site was not conspicuous (Fig. 10) except in follicles going through the terminal growth phase. Autoradiography has therefore revealed that the terminal growth phase is characterized by cortical deposition of the refractile bodies and a thickening of the vitelline membrane. At the beginning of chorion formation these structures add up to a total thickness of no more than 30 ,p, and in histological preparations they appear to be the only structures lying between the chorion and the outermost layer of trypan blue-stained yolk spheres. (The gap between the vitelline membrane and the chorion in Fig. 12 is an invariable artifact that arises due to

TERMINAL

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525

shrinkage of the yolk in terminal growth phase follicles during dehydration.) The volume increase of the oocyte due to elongation of its diameters by 60 p would be only 6%. Whatever its developmental function may be, therefore, cortical deposition is not a major component of the SO%volume increase occurring during the terminal growth phase. Collateral support for this conclusion is seen in the demonstration, which follows, that an important component of the volume increase is hydration of the oocyte.

Osmotic Changes and IVater Content during Terminal Growth The first clue to the possibility that hvdration contributes to the terminal growth phase was the demonstration of a substantial change during this period in the osmotic propertics of isolated yolk spheres. The protein yolk bodies of mature eggs have been shown to maintain their spherical shape when suspended in solutions of either 0.6 AP sucrose’ and 0.15 AI NaCl, or (Fig. 13) the osmotically equivalent 0.9 Af sucrose in the absence of ions (Telfcr, 1961); 1.4 11 sucrose leads to crrnation. while 0.6 JI .sucrose alone results in Ivsis, apparentlv bc-

Frcs. 13 and 13. Protein yolk spheres suspended in 0.9 M sucrose. In Fig. 14, the yolk spheres were from a follicle that had stained with trypan blue. while those in Fig. I .3 were from :I follicle that had initiatetl chorion formation. y 21X.

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cause the yolk spheres do not tolerate osmotic swelling. In preparing the earlier report, it was simultaneously observed that yolk spheres from immature follicles behave as though possessing a lower internal osmotic pressure. In a repetition of this test the yolk spheres of the most mature trypan blue staining cecropia follicles were found to be isosmotic with 0.6 M sucrose and crenated by 0.9 M sucrose (Fig. 14). When yolk spheres from successive follicles in single ovarioles were tested, the transition in apparent internal osmotic pressure from the equivalent of 0.6 M sucrose to 0.9 M was found to be completed during the terminal growth phase. The synchrony of this transition with the terminal growth phase suggested that osmotic swelling might account at least in part for the expansion of the oocyte. Accordingly, an effort was made to determine whether the dry weight of the oocyte keeps pace with its volume increase during the terminal growth phase. The oocytes of the largest staining follicles were loosened from their follicular envelopes by treatment for 5-10 minutes with 0.1% pronase in 10e3M KCl, 0.015 M MgC&, 0.004 M CaCl, and 0.18 M Tris-succinate buffer at pH 6.2, With higher values of potassium, the enzyme failed to loosen the follicle cells. When gently drawn into and out of a pipette with a diameter slightly less than the width of the whole follicle, the follicular epithelium along with the residues of the atrophied nurse cells could frequently be separated from the oocyte with the latter remaining intact. Separation was found to be facilitated by transferring the follicles to a solution in which 3% sucrose was substituted for the pronase. The dry weight of the oocyte in the mature egg was obtained by subtracting that of the hand-dissected chorion, from which the yolk and cyto’plasm had been removed by rinsing in dissecting solution, from the dry weight of the whole egg after ovulation. The materials to be weighed were transferred to tared aluminum foil vessels, blotted to remove residual dissecting solution, dried to constant weight at 110°C and equilibrated at room temperature for several hours over P,O, before weighing in a microbalance. The dry weights of 8 whole eggs ranged from 1.30 to 1.36 mg with a mean of 1.32 mg. Since 9 chorions from the same animal averaged 0.42 mg (with a range of 0.39-0.47 mg) , the mature oocyte dry weights were concluded to be close to 0.90 mg. Presumably due to difficulties entailed in the removal of follicle cells, the oocytes from the largest

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staining follicles yielded more variable results. The oocytes from 12 apparently successful separations yielded a mean value of 0.83 mg with a range of 0.67-1.00 mg and a standard error of kO.03. These data therefore suggest that the 50% volume increase during the terminal growth phase is accompanied by a dry weight increase of less than 10%. A major component of the terminal growth phase thereforr appears to be hydration of the oocyte. \it’hether an elevation in thr. internal osmotic pressure of the yolk spheres is in fact the driving force behind oocyte expansion and, if so, how it is generated remain to be solved, however. To summarize the observations described in this section, the period during which blood protein sequestering and protein yolk sphere production are important features of oocyte growth is followed by an approximately 24 hour phase during which water uptake plays an important role in producing a final 50% increase in the volume of the oocytcx. There is also a 10% increase in dry mass, however, and this must be due in part to the production of a new category of cortical bodies. These structures, which are substantially smaller and more refract& than the protein yolk spheres, readily label throughout the terminal growth period with tritiated histidine administered either by injection into the blood of the intact animal or in short term cultures. A thickening of the vitelline membrane between the oocyte and the follicle cells occurs simultaneously with refractile body formation. The terminal growth period generates the definitive shape of the oocvtc and terminates with the onset of chorion deposition.

Tnrnsformation of the Follicular Epithcliunl During vitellogenesis the follicle cells, which comprise a columnar epithelium surrounding each oocyte, are separated by 2-4 p-wide spaces that are filled with blood proteins (Telfer, 1961). Since this porous configuration arises at the outset of protein yolk deposition (e.g., Anderson, 1964, for Periplaneta), and disappears prior to chorion deposition (King and Aggarwal, 1965), it appears to be functionally related to blood protein uptake. The follicular epithelium might therefore be expected to undergo alterations simultaneously with the transition of the oocytye from vitellogenesis to the terminal growth phase. Investigation of the interactions between freshly dissected follicles and trypan blue in vitro in fact revealed two striking changes in the epithelium at the termination of blood protein uptake, a loss in

528

FIG. 15. 20 of adult

TELFER

Two chains of follicles development 3 hours

AND

ANDERSON

dissected simultaneously from a female on day after injection with trypan blue. Both chains

TERMIKAL

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529

penetrability to trypan blue, and a drastic reduction in a capacity for extracellular dye binding observed in vitellogenic follicles. Demonstration of these changes was initiated by observations such as those illustrated in Fig. 15. The lower chain of folIicles is the same as that pictured in Fig. 1. It had been removed from the animal 3 hollrs after trypan blue injection and, in the interests of clarity, dissected free of the ovariole wall. The upper chain of follicles had been removed from the animal simultaneously, dissected from its ovariolc wal1, and then immersed in 1% trypan blue of the same ionic composition and pH as that described earlier for the dye injection experiments. After removal from the dye I5 minutes later, *it was rinsed repeatedly in dissecting solution until the washings no longer contained visuallv detectable trypan blue. As can be seen, follicles in the same size range as those that had sequestered dye from the blood in the animal no\\bound it with substantially greater intensity. Freshly dissected ovarioles from uninjected animals have been repeatedly found to respond in the same manner. Indeed, this method appears in all regards to be as rcliablr as trypan blue injections for identifying the stages of follicnlar development that sequester blood proteins. Histological analysis of follicles stained in ~jifro indicated that the dye was bound in all extracellular spaces and membranes adjacent to the follicular epithelium ( Figs. 16, 18. and 19), including the brush horder at the surface of the oncyte, the g-3 /,.-thick basal lamina covering the outer surface of the follicle (the tunica propria of Ronhag, 1958)) and the spaces lying between follicle cells. Therefore, the stages which are able to deposit trypan blue along with proteins from the blood in the protein yolk spheres of the oocyte are also able to bind the dye with apparently great intensity in the e~tracellular spaces of the follicle. Estracellular dye binding \vas not apparent after injection

were removed from their ovariole walls, after which the ripper one‘ was bathctl in 1% trypan blue for 15 minutes and then rinsed in dissecting solution. x3.2. FIGS. 16 and 17. Two follicIes from the same olariole that had been simultaneonsTy cq~osrd to 1% trypan blue in citro and then rinsed in dissecting solution nntil the washings were colorless. The extracellular spaces of the follicle in Fig, I6 had been deeply stained, along with the basal lamina and the surface of thp oocyte. A few small yolk spheres are also stained. The follicle in Fig. 17 showed the light staining characteristic of R follicle jllst tsntvrillr,r thr terminal growth pbasc. Ronin’s fixation. X ,543.

530

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of the dye into the animal, owing in part to the lower concentrations used, and in part, as will be shown in a later paper, to competition with blood proteins for the extracellular binding sites, Equally striking in these experiments was the loss of trypan blue binding capacity in follicles that had become large enough to enter the terminal growth phase (Fig. 15). In histological preparations the basal lamina of such follicles appeared lightly stained (Fig. 17), but the amount of dye in the intercellular spaces was greatly reduced, and confined to the outer or basal three-quarters of the epithelium. The configuration of the spaces (see also Fig. 8) and the distribution of dye suggested that neighboring follicle cells had come together to exclude the dye from the inner quarter or third of the intercellular spaces, and from the oocyte surface, whose brush border now contained no detectable trypan blue. A similar configurational change was observed in low power electron micrographs by King and Aggarwal (1965). To the cessation of blood protein uptake, hydration of the oocyte, thickening of the vitelline membrane, and the production of refractile bodies in the oocyte cortex, therefore, the establishment of an excluding zone in the follicular epithelium, and the loss of an extracellular trypan blue binding capacity may be added as part of the complex of changes occurring in the 2-mm follicle. The loss of dye-binding ability reflects a fundamental change in the extracellular environment of the follicle cells. It is tempting to speculate that dye binding is effected by an extracellular adsorbant, which the folhcle cehs cease to secrete at the termination of bIood protein uptake. Before reaching this conclusion, however, it was necessary to rule out the possibility that trypan blue binding might be due to coprecipitation with the blood proteins that are abundant in the spaces during vitellogenesis. This possibility was tested by exposing the follicles to a variety of conditions that might be expected to remove most of the blood proteins from the spaces, and then observing their dye-binding ability. It was found during this investigation that, while the dye-binding property is lost from the spaces during exposure to dissecting sohrtion at pH 6.3 and higher, it is maintained intact for at least an hour in the same solution buffered with Tris-succinate at pH 5.5. Blood proteins on the other hand rapidly moved from the extracellular spaces of the follicle into the external medium at both pH values. In a representative experiment, part of one ovariole containing about

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35 vitellogenic follicles was removed from a l7-day developing adult. rinsed briefly in pH 5.5 dissecting solution to remove blood adhering to the surface of the ovariole, and subjected to 4 consecutive Sminutc’ washes in 0.15 ml of the pH 5.5 solution at 3°C. The follicles were s~lbsequcntly placed in the 1’s trypan blue solution (pl1 6.3) descrih(>d

follicle stained irr Frc:. 18. Tangential section, 10 ,U thick, of a vitellogenic citro wit11 trypan blue. \Vhile section thickness and angle resulted in Sony 01 (‘I’lapping of successive layers, the staining of the outmnost lay, the hasal larnir~a, and of the intercellular spaces is particularly clear. Bouin’s fixation. x218. Fx. 19. Two follicles from a 17-clay developing adult after staining with trvpan b111(~for 1 hour. The follicle on the left had been transferred dircctl) from the animal to the dye solution. That on the right had hecn exposed for 1 hollr to dissecting solution at pH 5.5 in order to soak out tliffnsihl~~ blood proteins prior to staining. Rorlin’s fixation. X 545.

above, incubated in the co1.d;for 1 hour, and then washed Tvith cold, pH 55 dissecting s+itkn until no further free trypan blue wils released. Comparison of the trypan blue staining in sections of Houin’s fixed follicles treated in this way with that of follicles transferred from the blood directly into trypan blue showed a close similarity (Fig. 19 ). Analysis by the Oudin method of the four bvash solutions for concc,ntrations of the two major vitellogenic blood proteins indicated that

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these proteins diffuse readily from the extracellular spaces into the external medium, In the first wash the female and carotenoid proteins were, respectively, 0.46% and 0.55% their concentration in the blood. The subsequent 3 washes contained only traces of the two proteins too low to be measured by the Oudin method. Estimates, from histological preparations, of the volume of the interfollicular cell spaces indicated that the amounts of the two proteins in the wash were at least sufficient to fill the spaces at concentrations equivalent to those in the blood. In order to determine whether some blood proteins might have spontaneously precipitated in the extracellular spaces at the low pH, the blood of a developing female was dialyzed against the pH 5.5 dissecting solution. No blood protein precipitation could be detected. The retention of trypan blue between the follicle cells therefore indicates in all probability an extracellular or cell surface material, which either binds trypan blue directly or combines with a blood protein which in turn reacts with the dye. Although several functions for this product can be imagined, its restriction to those stages which sequester blood proteins hints that its function is intimately related to the uptake process. In any event, the sudden, time-specific loss of the dye-binder confirms a close physiological, as well as morphological, integration of the changes occurring in follicle cells and oocyte during the transition from vitellogenic to postvitellogenic growth. DISCUSSION

The cessation of blood protein uptake and the initiation of the terminal growth phase have been shown to entail an extensive complex of changes. Every property thus far examined appears either to be lost or to undergo an abrupt acceleration in the 2-mm follicle, and the list will presumably lengthen with time. While the interrelations between component changes of the terminal growth phase are not at once obvious, it is hardly conceivable that events as fundamental to the physiology of cells as hydration, for instance, would not have a profound impact on the system as a whole, and thus, however indirectly, affect many follicular activities. The follicle may therefore be thought of as undergoing at this time a change of state that has many manifestations in its structure and function. The component changes thus far recognized are most easily related for the present to the cessation of blood protein uptake which domi-

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nates vitellogenesis in each cecropia follicle for almost a week. The closing of the intercellular spaces in the follicular epithelium, and the loss of the extracellular adsorbent detectable with trypan blue are plausibly related in a direct way, perhaps even causally, to the cessation of blood protein uptake. The thickening of the vitelline membrane and the production of the refractile bodies in the oocyte cortex must reflect either new dispositions of histidine labeling materials which were entailed at an earlier stage in the uptake of blood proteins, or a change in the synthetic and secretory activities of the follicle cells 01 the oocyte. One possibility that merits future testing is that the oocvtc, surfacc~, upon being denied access to blood proteins, continues to generatc’ membrane-limited structures, now in the form of the refractilc I)otlics rather than yolk spheres, from pinocvtoticallv derived product\ of follicle cell secretion. Thr relation of hydration to the termination of blood protein uptakcl is Icss obvious, but even here a number of possibilities exist. For iu,. stance, pinocvtotic yolk deposition with its attendant devices for concentrating some blo’od proteins and excluding others may leave the oocyte in a relatively dehydrated condition. Since the opportunity for frcclv/ diffusing extracellular proteins to contaminate the yolk will 1,~ directlv proportional to the fluid volume of the pinocytotic vesiclrs. a highly selective mechanism would entail as small a volume as possiblr for a given amount of surface area (e.g., Telfer. 1961; Hoth and Porter, 1963 ). This situation in conjunction with the fact that a clcitloic egg such as that of cecropia, which is laid in an exposed fashion on tlica surfac~e of a leaf, must have a significant vv2~trlr coiiservation prob I(sni, mav cnngeuder the necessitv for thca sI)cGil p(~riotl of hvdratioii S(YWin thci terminal growth phase. Finally, before the apparent increase iu internal osmotic pressim of the yolk spheres can be plausibly related to other changes, the question of vvhcthrr it is generated by the addition of nevv solutes to the yolk spheres, or by the chemical dissociation of precsisting hydrophilic components, will have to be answered. \\hatevrr the interrelations between component changes may be. t11e ovc,rall transformation of the 2mm follicle is most probably genclrated and controlled from within the ovary itself. One follicle in each ovariolcl enters the terminal growth phase every 4-f-5 hours from da\. 19 of adult dcvelopmcmt until after the emergence of the moth on tlav 2.3 ( Tclfer and Hutberg, 1960). The nutrient. physical. and, if rel-

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evant, hormonal properties of the blood necessary for this phase of development must therefore be continuously adequate for at least 4 days. The 20-30 vitellogenic follicles that have not accumulated an adequate complement of yolk on day 19 of development remain refractory to these conditions, however, until each in turn is 2 mm long. Thus the stage of its development or its po’sition in the ovariole, rather than the sudden availability of a crucial factor in the blood, must trigger the entry of each follicle into the terminal growth phase. Because of a regrettable scarcity of literature on the developmental physiology of oocytes in the final phases of egg formation, it is not possible to know which of the several aspects of the terminal growth phase are unique adaptations of cecropia or of moths, and which, if any, are more general characteristics of oocyte development. The terminal growth phase can fruitfully be thought of at the present time, however, as an early stage in the conversion of the oocyte from vitellogenesis to embryogenesis. Since 45 days elapse between the initiation of chorion deposition and the time the egg is inseminated and laid by the moth, a substantial proportion of the period of conversion remains to be analyzed. SUMMARY

Vital staining with trypan blue was found to provide a simple and reliable index to the stages of follicular development that sequester blood proteins in yolk spheres. It was thus possible to show that blood protein uptake terminates about 24 hours before chorion formation begins, when the oocyte has reached no more than two-thirds of its final volume. The ensuing size increase, which appears to result primarily from hydration, is accompanied by an extensive set of transformations in follicular function. These include closure of the intercellular channels across the follicular epithelium, the loss of an extracellular material that binds trypan blue and may be involved in blood protein uptake, a thickening of the vitelline membrane, the production of highly refractile, spherical bodies in the oocyte cortex, and an apparent increase in the internal osmotic pressure of the yolk spheres. The changes are interpreted as components of an early step in transforming the oocyte from a vitellogenic system to a mature egg. We would like to acknowledge the able technical assistance of Mrs. Elizabeth Wallace. The sections shown in Figs. 7 and 8 were generously supplied by Dr. Barbara Stay, and the culture experiment (Fig. 9) was based on the experimental background work and assistance of Mr. Steven Hausman.

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REFERENCES E. (1964). Oocyte differentiation and vitellogenesis in the roach, PC,)iplaneta umericanu. J. Cell Biol. 20, 131-155. HIEH, K. ( 1963). Autoradiographische Untersuchungrn iiber die Leistungcn ties Follikelepithels und der Nahrzellen bei der Dotterbildung untl Eiwrissynthesc im Fliegenovar. Arch. Entwicklzmgsmech. Orgal. 154, 552-575. RONHAr;, I’. F. ( 1958). Ovarian structure and vitrllor$nesis in insects. Atrtt. fhji Entortd. 3, 137-160. FEDEn, N., and SIDMAN, R. 1,. ( 1958). ~lethotls and principlrs of fixation l)\ freeze-sllbstitution. J. Biophys. Biochem. C!ytol. 4. 5R.T602. RINC, R. C., amtl AGGAHWAL, S. K. ( 1965 ) Oogenesis in H!/“!o!~/forcl WCIY~/J~U. Growth 29, 17-83. E. A. ( 1963). Studies of ovarian follicle cells 111 rht:, R. c., and Kocx, llrosophiZ~4. (hcurt. J. Jficnwop. Sci. 104, 297-380. analysis of blood protcirr iiptak~~ \IEI.IUS, 11. E. ( 1966). An autor;itliojiraphic ;wd protein yolk sphere formation bv cecropia nioth oocvtcs. Ph.D. thesis, Uni\ersitv of Pennsylvania. ( 3rd ed. ), pp. 202, 282-283. I’AHKEH, IL’C. ( 1961 ). “Methods of Tissiie Culture” Harper, New York. PI~EEH, J. R., and TELFER, W. H. ( 1957). Some clfrcts of nonreacting sribstaiices in the quantitative application of gel diffusion tcchnicliir~s. J. Inr nrntrol. 79. 288-293. RAhrAhrunrv, 1’. 5’. (1964). On tl le contribution of the follicle cpithelium to tlrc, deposition of volk in the oocvtc of Panorpcl co~tun~~~~is. l:‘.vptl. Cd/ Rc.s. :<:I. 601-604. ’ b’l’H, ?‘. F., and PORTER, k’. R. ( 1964). Yolk protcG uptakes iii the or)cvte of tllc, moscluito Acdc~s aeg!y@ (L.). J. Cell Rio/. 20, 313-332. STAY, 13. (1965). Protein irptake in the ooc!tes of the ccyrcqii;c rirotli. 1. role of a sex-limited female protein in egg formation ln, the cccropia silkworln. J. Cm. Ph!/siol. 37, 539-558. ‘I‘ELFEI{, W. H. ( 1960). The selective accumulation of blood proteins bv tlic oocytes of saturniid moths. Bid. Bull. 11’8, 338-351. TELFEH, \V. H. (1961). The route of entry and localization of blood proteins in the oocytes of saturniid moths. J. Biophys. Biochern. C!/toZ. 9, 747-759. TELFEH, IV. II. ( 1965). The mechanism and control of volk formation. ~rrrt. R~I-. Erctortto~. 10, 161-184. ‘I‘IXFEH, I\‘. H., and RUTBEIK, L. I). ( l$MI ). Tlrc eflwts of t,]oo(l I)r()tpill (jeplt.. tiou on the ,qowth of the oocytes in the cecropia nroth. Hiol. Brrll. 118, 3.52-366. TELFE~~, \t’. If., and \VILLIAMS, C. hf. (195.3). I mmunologicnl studies of insect metamorphosis. I. Qualitative and quantitative changes in the blood proteins of the cecropia silkworm. J. Gw. Ph!ysiol. 36, 3891113. ANDEHSOS,