A correlative microscopical analysis of differentiating ovarian follicles of mammals

A Correlative Microscopical Analysis of Differentiating Ovarian Follicles of Mammals

EVERETT ANDERSON, RICHARD F. WILKINSON,2 GLORIA LEE AND SAMUEL MELLER Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Haruard Medical School, 45 Shattuck Street, Boston, Massachusetts 021 15

ABSTRACT The mammalian ovary has been studied by optical microscopy and by scanning and transmission electron microscopy with the purpose of pre- senting an integrated view of the differentiating mammalian follicle. During follicular development, changes in the granulosa cells are particularly notewor- thy and include dramatic modifications in cell shape coincident with antrum formation. The cytoplasmic processes of those granulosa cells immediately sur- rounding the oocyte, as well as the more peripheral granulosa cells comprising a second and third layer, traverse the zona pellucida, infrequently interdigitate with the microvilli of the egg, and make both desmosomal and gap junction contacts with the oocyte. The zona pellucida is thus distinguished by numerous fenestrations of varying diameters. The membrana limitans (basal lamina) is a bipartite structure composed of (a) a homogeneous stratum upon which the peripheral layer of granulosa cells rests, and (b) an outer region of collagen-like fibers. The specific advantages and limitations of the different methodologies utilized to study folliculo-genesis are discussed.

The egg occupies such a unique position within the life history of many organisms it was not surprising that with the development of electron optics, together with the applica- tion of appropriate methodology for its use in biological investigations, numerous investigators were prompted to turn their energies to the study of the finer anatomy of this impor- tant cell type. (See Anderson, '74; Hertig and Barton, '72; Zamboni, '70). The purpose of the present study is to extend these observations with the hope of adding further information to the overall structure of the developing mammalian ovarian follicle.

MATERIALS AND METHODS

Animals Mice from the CD-1 strain and New Zealand

white rabbits were used in the studies to be described below. Following anesthetization with dibutal, the ovaries were removed and placed in fixative.

Microscopy Whole ovaries from mature mice were fixed

for 45 minutes in a fixative containing 3% glu- taraldehyde, 1% paraformaldehyde, and 5% sucrose in 0.1 M sodium phosphate buffered to

J. MORPH. (1978) 156: 339-366.

pH 7.4. Ovaries from immature mice were sim- ilarly fixed except that the sucrose was omit- ted. Non-stimulated and sexually stimulated (3, 5, 7, 9, 11, and 12 hours post-coitus) rabbit ovaries were fixed for 1.5 hours with 3% glu- taraldehyde containing 2% sucrose buffered to pH 7.4 with 0.1 M sodium cacodylate-HC1.

Following primary fixation, some of the ovaries were hardened by immersion in a l% solution of thiosemicarbazide (TSC) that had been dissolved in either 0.1 M sodium phosphate (mouse) or 0.1 M sodium cacodylate-HC1 (rabbit) each buffered to pH 7.4 (Anderson et al., '77). The specimens were kept in this solution for about 30 hours a t 25°C. Following this step, each ovary was broken in various planes by gentle but continuous pressure from a scalpel blade. In some studies, especially on vascularization of the theca interna, the fixed, but non-TSC-treated ovaries were transected with a razor blade and processed for SEM (see below).

The prepared tissue was then rinsed several I This investigation was supported by Grants (HD 06822; HD

06645) from the National Institutes of Health and the United States Public Health Service.

Dr. Wilkinson's present address is: Department of Biological Sci- ences, Allen Hancock Building, University of Southern California, Lo8 Angeles, California 90007.

339

340 E. ANDERSON. R. F. WILKINSON. G . LEE AND S. MELLER

times with buffer and subsequently post-fixed for 1 hour in 1% osmium tetroxide buffered to pH 7.4 with either 0.1 M sodium phosphate (mouse) or 0.1 M sodium cacodylate-HC1 (rabbit). A graded ethanol series was used to dehy- drate the tissue, followed by three changes of amyl acetate. The tissues were then critical point dried through carbon dixode using a Samdri PVT-3 apparatus (Anderson, ’ 5 6 ) . The dried specimens were mounted on SEM stubs and coated with gold-palladium, using a Tech- nics Hummer instrument (Alexandria, Vir- ginia), or coated in a Denton Vacuum DV-502 evaporator equipped with an omnirotating stage. All specimens prepared for SEM were examined with either an ETEC autoscan,3 JEOLCO-US or JSM-35 scanning electron microscope.

In an effort to study some aspects of ovulation by “conventional” SEM in the rabbit, 10 animals were mated and following 3, 5, 7, 9, 11, and 12 hours post-coitus, two animals from each time period were anesthetized with sodium pentobarbital, the ovaries removed and fixed for 30 minutes i n the aldehyde fixative (see above). Subsequently, the large pre-ovulatory and ovulatory follicles were excised and allowed to fix further for one hour. After primary fixation a portion of the aforementioned tissue was post-fixed for one hour in 1% osmium tetroxide and dehydrated in a graded series of ethanols. The specimens were processed for SEM as indicated above.

Several pieces of tissue from the study dealing with ovulation, after having been examined in the SEM, were semi-thin and thin-sec- tioned after embedding in Epon-Araldite, according to the procedure of Meller et al. (‘73).

Some ovaries were cut into small pieces and processed for light and transmission electron microscopy as described by Anderson et al. (’76); some pieces were impregnated with lanthanum, while others were processed for freeze-fracture according to procedures reported previously (Albertini and Ander- son, ’74).

In an effort to stain matrix components of the basement lamina of the ovarian follicle, some tissue was fixed in a n aldehyde fixative containing 2% tannic acid (Van Deurs, ‘75) and processed for transmission electron microscopy (TEM) (see above).

OBSERVATIONS AND DISCUSSION

With increasing use of the scanning elec-

tron microscope as a powerful analytical tool in reproductive biology and with the advent of methods designed to utilize this instrument effectively in research, we feel tha t a clarifi- cation of technical nomenclature should be made. Authors have used various methods to dissect tissues so tha t optimum surface de- tails of cells can be retained. Included among these techniques are freezing in liquid ni- trogen (Humphreys e t al., ’74) and blunt dissection (Miller and Revel, ’75). All of these methods, including ours, are designed to harden the tissue before dissection. Rather than utilize the terms “breaking,” “fractur- ing,” or “cracking,” and thus to avoid confu- sion of these techniques with the freeze-fracture methodology, we suggest tha t the term “dissection of hardened tissue” or simply “HTD” be used to describe any scanning electron microscopical technique, such as the one we present here, where thiosemicarbazide (TSC) was used.

The results using TSC to harden the ovary prior to dissection proved to be simple to use and repeatable. With care, one could consist- ently make preparations such as the one pre- sented in figure 1. Here the complementary halves of the ovary were mounted so that a more complete study of each follicle could be made. As shown in figure 1, the ovary is covered by the ovarian epithelium, which is continuous with the peritoneum (Anderson e t al., ’76). Histologically, the ovary is divided into a central medullary area, containing vascular and some connective tissue elements, and a cortical region consisting primarily of follicles in varied developmental stages that reflect the degree of follicular maturation. Here i t may be pointed out tha t regardless of the time in the cycle, or even during pregnan- cy, one can always find follicles in different stages of development. This fact is consistent with the observation tha t mammalian oogen- esis is a continuum upon which is superim- posed the FSH and LH surge for ovulation (Peters e t al., ’75; Richards and Midgley, ’76; Greenwald, ’74).

General features of the ovarian follicle In a n effort to facilitate the ease of pre-

sentation and integrate our correlative find- ings, figures 2 and 3 call attention to the prin- cipal features of the anatomical and physio-

3 W e are grateful to Doctor L. V Leak, Department of Anatomy, Howard Medical Schwl, for the use of the ETEC autoscan, scannlng electron microscope.

OVARIAN FOLLICLES OF MAMMALS 341

logical compartments of an ovarian follicle. Each of these will, in turn, be discussed below. The oocyte is encompassed by two non-cellular boundaries: (1) the zona pellucida and (2) the membrana limitans (basal lamina). Between these two is a cellular stratum, the granulosa layer. During the ontogeny of the ovarian follicle, a fluid-filled cavity known as the antrum appears among the granulosa cells. Circum- ferentially arranged outside of the membrana limitans are two functionally different cellular layers (fig. 2) . The first of these is the vascularized theca interna whose cells secrete steroids. Second is the theca externa, composed of fibroblast-like cells, the function of which is unknown.

Pre-antral follicle The early pre-antral follicle is initially sur-

rounded by a single layer of squamous-shaped granulosa cells (inset, plate 3). In our scanning micrographs of these early follicles only the surface of granulosa cells is revealed, showing no distinguishing features. As these follicles continue their differentiation, the squamous granulosa cells become cuboidal or low columnar in shape. In the scanning micrograph illustrated as figure 5, the oocyte has been transected. Other than the unidentified spherical bodies there is little intracellular information relative to the oocyte when one compares such an image with what has been obtained with light microscopy and TEM (Hertig and Barton, ’72; Anderson, ’74) and by freeze fracture methodology (Anderson et al., ’77).

During early development, when the follicle is encompassed by a single layer of squamous granulosa cells, a thin granular homogeneous basal lamina subtends these cells. In growing follicles whose granulosa cells are cuboidal in shape, an additional fibrous component of the basal lamina is visible. In figure 6, the artifactual displacement of the theca interna from the granulosa cell layer permits an appreciation of the fibrous layer of the basal lamina. Sections of materials preserved with tannic acid-containing fixative disclose the transverse periodicity of these fibers. As shown in figure 7, the collagen-like fibers are intensely stained with tannic acid. These observations prompt the suggestion that the basal lamina of the ovarian follicle is a bipartite structure, the inner portion being the granular homogeneous component presumably derived from the peripheral gran-

ulosa cells, and the other being a colla- genous component which conceivably origi- nates from fibroblast or fibroblast-like cells that can be found among the stereoid-secret- ing cells in the theca interna.

As alluded to above, the outer layer of granulosa cells presumably is the only one to contribute to the inner granular homogeneous layer, which comprises the membrana limitans. When one examines the population of granulosa cells of pre-antral and an t ra l follicles, no intercellular matrix containing abundant glycosaminoglycans is usually found between these cells. However, in follicles of some mammals, dense accumu- lations of a homogeneous material may be found among granulosa cells (fig. 41, which are referred to as Call-Exner bodies (Call and Exner, 1875) . I t is difficult or nearly impossible to identify the Call-Exner bodies with the SEM technique. When seen with TEM the components of the Call-Exner body are separated from each associated granulosa cell by a space giving the appearance of a continuous basal lamina. I t is conceivable that certain granulosa cells produce the material comprising these bodies, which, as seen in figure 4, are composed of a reticulated homogeneous continuum to which is associated some fine dense filamentous material. The significance of this apparent functional difference among granulosa cells during follicle differentiation is unknown.

As the follicle advances in its development, some of the granulosa cells become polyhedral in shape, while others become markedly elongated (figs. 2 and 5 , in contrast to fig. 12; also see Chang et al., ’77). Many of the cells (fig. 12) develop exaggerated pseudopodial processes and become flask-shaped. The long cytoplasmic processes often arise from cells that are located in the second and sometimes the third “layer” of granulosa cells peripheral to the oocyte. This latter fact has been unappreciated by TEM.

Zona pellucida Between the oocyte and the encompassing

layer of granulosa cells is an accellular stratum, the zona pellucida. The HTD technique employed here reveals the zona pellucida to be fenestrated by numerous variously sized pores (fig. 8). The larger fenestrations are interpreted to be those areas where broad pseudopod-like granulosa cell processes con- tact and/or traverse the zona pellucida (fig. 81,

342 E. ANDERSON. R. F. WILKINSON. G. LEE AND S. MELLER

large arrow). Conversely, the smaller pores tha t punctuate the zona are those regions where smaller processes from granulosa cells traverse the zona (fig. 8, small arrow). These relationships can also be fully appraised in conventional TEM (fig. 91, in which portions of dark staining granulosa cell cytoplasmic processes can be distinguished from the more electron-lucid microvilli of the oocyte.

There is a n agreement concerning the porous nature of the zona pellucida (Flechon e t al., '75; Dudkiewicz e t al., '76). However, not all investigators are in agreement with re- spect to the structural organization of this stratum. TEM reveals t ha t the zona pellucida consists of a rather electron-opaque, fine filamentous material (fig. 9). In our SEM images of the zona pellucida, we found it to be composed of many globular units of different di- mensions (fig. 8). By use of SEM, Dudkiewicz e t al. ('76) reported tha t the zona pellucida of hamster oocytes was composed of a complex network of interconnecting fibers. From our studies we are unable to report a similar structural organization. What this difference in architectural design may represent is unknown. Perhaps i t is worthwhile to point out tha t Dudkiewicz, e t al. ('76) subjected their eggs to 0.1% hyaluronidase for 7-10 min at 20- 22" C before preparing t,hem for SEM. The observed structural variations of the zona may be a result of differences in precipitation or of species differences.

The precise origin of the zona and its function prior to ovulation are poorly understood (Modlinski, '70). In terms of i ts derivation, some investigators report tha t i t is a product of the oocyte (Hartman, '26; Sotelo and Porter, '59; Odor, '60; Kang, '74); some advise tha t i t is the product of the granulosa cells (Trujillo-Cenoz and Sotelo, '59; Hertig and Barton, '72); while still others report t ha t i t is the product of both (Wartenberg and Stegner, '60; Zamboni, '70). Almost all histochemical studies made to date indicate tha t the zona is rich in polysaccharide (Silva Sasso, '59) and contains negatively charged sialic acid (Sou- part and Noyes, '64).

Antral follicle With the "HTD" technique employed for

one of our SEM techniques we found on other- wise smooth-surfaced granulosa cells disk-like regions displaying either a raised ragged surface or a shallow depression (fig. 12). We be- lieve tha t these disk-like areas are isomorphic

with gap junctions. The 3-4 nm of the gap junction is accentuated in tissue permeated with lanthanum (fig. 12, inset A). By use of the freeze-fracture technique, the supra-mo- lecular organization of the gap can be seen in figure 12, inset B. Here the aggregate of parti- cles forms a domain which is nearly disk- shaped. I t should be pointed out tha t not only do gap junctions appear between granulosa cells, but they are also found between the oocyte and granulosa cells (Anderson and Albertini, '76; Anderson et al., '77). Experi- mental evidence suggests tha t the physical basis for communication between animal cells is the gap junction (Bennett, '73; Gilula, '73; Furshpan and Potter, '68). In connection with the presence of gap junctions between the cellular population of the ovarian follicle, we suggested tha t they may not only ". . . partici- pate in the preantral growth of the female gamete, but also serve to regulate nuclear events associated with meiosis before and at the time of ovulation." (Anderson and Alber- tini, '76).

Antrum When the growing follicle in vivo becomes

dependent on gonadotrophins, there appear among the follicle cells fluid-filled areas tha t eventually become confluent with each other to form the antrum, a cavity filled with secre- tion from the follicle and exudates from the plasma (Hisaw, '47; Edwards, '74).

In the SEM images, the antrum appears as a relatively smooth-walled cavity during i ts ini- tial formation (figs. 10, 13). However, the antrum of the preovulatory or Graafian follicle is filled with a rather solid-looking bolus (fig. 141, whose appearance is probably the result of protein denaturation in the antral fluid during fixation. Within the matrix of this solid appearing mass, one can discern some cellular elements (fig. 14, arrows) tha t may be granulosa cells comprising t h e cumulus oophorus.

Theca Very early during the growth of the ovarian

follicle the theca interna develops as an aggre- gation of cells adjacent to the membrana limitans. As the theca interna develops, these cells become glandular and are involved in the production of estrogen (Ryan e t al., '68). More- over, the theca interna becomes richly vascularized (fig. 10) and at the time of ovulation its capillaries become fenestrated (Byskov,


'69). We were interested in defining the changes in the vasculature of the theca interna during the ovulatory process and had hoped that the HTD technique would be a useful procedure. However, we found the method un- suited to the study of the vascular elements due to the lack of adequate definition in both thecal and stromal elements of the ovary. Therefore, we modified our technique by simply cutting fixed but non-thiosemicarbazide- treated ovaries with a razor blade. As a result the appearance of all blood vessels was en- hanced, as illustrated in figures 10 and 11. Blood vessels of various sizes are shown a t the arrows. At higher magnification, certain vascular elements of the theca interna are clearly defined (fig. 11). The granulosa-cell layer is oriented toward the lower portion of the micrograph. The endothelium of a small vein and the capillaries containing erythrocytes are also shown (fig. 11).

I t is interesting to point out that in another study we found changes in the tight junctions between endothelial cells of capillaries in the theca interna following sexual stimulation in rabbits (Anderson e t al., '74). In this study it was shown that the tight junctions in non- stimulated animals were composed of a few in- terconnected linear elements in association with the gap junction. After sexual stimulation there was an increase in the number of in- terconnected linear elements and they ap- peared to anastomose with each other. The functional significance, if any, of the capillaries of the theca interna becoming fenestrated and the increase in tight junctional elements prior to the ovulatory process is unknown. It is conceivable that this change is in anticipation of function of the corpus luteum.

Preovulatory or Graafian follicle Prior to ovulation the cells of the ovarian

epithelium are rather cuboidal and, when seen with the SEM, they are adorned with numerous microvilli. When the ovulatory process is induced in the rabbit by sexual stimulation, one notices over a period of time numerous presumptive ovulatory points in the form of swellings on the ovarian surface. For literature dealing with the mechanism of ovulation the reader is referred to the following papers: Lipner, '73; Espey, '74; Schwartz, '74; Bjers- ing and Cajander, '74; Blandau and Rumery, '63. Relatively little significant change in the morphology of the cell over these presumptive

ovulatory regions occurs during 3 , 5 or 7 hours post-coitus. Subsequent to the aforementioned times, one notices distinct morphological changes of the ovarian epithelial cells over these dome-shaped presumptive ovulatory areas. These cellular changes are the prelude to the formation of the stigma. In an effort to discuss these cellular changes, we have for convenience of description, as have others, divided the large pre-ovulatory or Graafian follicle (fig. 15, 12 hr post-coitus) into three regions: (1) apical, (2) intermediate, and (3) basal (cf. Nilsson and Munshi, '73; Bjersing and Cajander, '74). The cells on the apex become extremely flattened with relatively smooth surfaces and very few microvilli (figs. 16, 17). There are many intercellular spaces, which suggest that the apical cells become uncoupled from each other. In some areas, the exfoliation is complete, thereby making visible the underlying collagen, one component of the tunica albuginea (fig. 16).

As one moves down over the lateral aspect of the apical zone, one encounters a morphologi- cally distinct population of cells comprising the intermediate zone. The cells of the intermediate zone appear dome-shaped; the dome is studded with short, stubby microvilli, whereas the remaining portion of the cell has rather long microvilli (fig. 18).

The cells of the basal zone are rather cuboidal and are entirely covered with rather long, slender microvilli (fig. 19). I t is obvious that the cells of the apical zone are lost prior to and during ovulation and the ones of the intermediate and basal zone are retained. The functional significance of the differential size and distribution of microvilli between cells comprising the intermediate and basal zones is unknown.

We examined the ovary of a rabbit 12 hours post-coitus and figure 20 is the SEM of a portion of what we initially identified as an ovulatory region. A closer inspection of the constituent cells revealed them to vary in size, shape, and number of microvilli (fig. 21). The variation in size and shape of these cells is what one would expect in the granulosa cells comprising the cumulus oophorus; and these cells were so identified. However, when the ovulatory follicles were fixed for the HTD procedure, i t was noted that the antral fluid became coagulated to form a solid bolus. We therefore decided to section that same material from which the scanning micrograph in figure 20 had been made, with the hope of

344 E. ANDERSON, R. F. WILKINSON. G. LEE AND S. MELLER

revealing an enclosed oocyte. The results of this procedure are shown in figure 22. The structure is presumably a papilloma similar to that observed by Bjersing and Cajander ('74). This observation indicates tha t where the identity of tissue processed using HTD technique or other SEM techniques is uncertain, these same specimens should be subsequently embedded for either light or transmission electron microscopy for positive identification (Meller et al., ' 7 3 ) .

While the purpose of utilizing the HTD procedure was to examine ovarian tissue in situ, it became obvious tha t this technique is not suitable for studying the ovulatory follicle. As illustrated in figure 23. accompanying the release of the oocyte and companion cumulus cells at ovulation not only the oocyte with associated cumulus cells is released but also antral fluid and suspended granulosa cells. As a result of fixation the coagulated antral fluid obscures recognizable cellular features.

Thus i t becomes evident tha t while a combination of methodologies has permitted a n integrated view of folliculogenesis, this same combination has not extended our knowledge of the ovulatory process and therefore new methodology or a refinement of old techniques must be introduced.

LITERATURE CITED

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Anderson, E. 1974 Comparative aspects of the ultrastructure of the female gamete. Internatl. Rev. Cytol., (Suppl. 4): 1-70.

Anderson, E., D. F. Albertini and R. Letourneau 1974 The ultrastructural design of the theca interna during the ovulatory process in the rabbit. J . Cell Biol., 63: 8, abstract.

Anderson, E., and D. F. Albertini 1976 Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J . Cell Biol., 71: 680-686.

Anderson, E., D. F. Albertini and R. F. Wilkinson 1977 Cy- tological differentiation of the female gamete. In: Inter- national Cell Biology 1976-1977. B. R. Brinkley and K. R. Porter, eds. The Rockefeller University Press, New York, pp. 561-568.

Anderson, E., G. Lee, R. Letourneau, D. F. Albertini and S. M. Meller 1976 Cytological observations of the ovarian epithelium in mammals during the reproductive cycle. J . Morph., 150: 135-166.

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Call, E., and S. Exner 1875 Zur Kenntnis der Graafischen Follikels und des Corpus Luteum beim Kanninchen. Sitzungsber. Math.-Naturwiss. K1. Kaiser, Akad. Wiss. Wien, 71: 321-328.

Chang, S. C. S., W. Anderson, J . C. Lewis, R. J. Ryan and Y. H. Kang 1977 The porcine ovarian follicle. 11. Electron microscopic study of surface features of granulosa cells a t different stages of development. Biol. Reprod., 16: 349-357.

Dudkiewicz, A. B., C. A. Shivers and W. L. Williams 1976 Ultrastructure of the hamster zona pellucida treated with zona precipitating antibody. Biol. Reprod., 14: 175-185.

Edwards, R. G. 1974 Follicular fluid. J. Reprod. Fert., 37: 189-219.

Espey, L. L. 1974 Ovarian proteolytic enzymes and ovulation. Biol. Reprod., 10: 216-235.

Flechon, J., S. Berstrom, S. Jaszczak and E. S. E. Hafez 1975 Techniques for critical point drying of gametes and embryos. In: Scanning Electron Microscopy. 0. Jahani and I. Corvin, eds. ITT Research Institute, Chicago, pp. 325-331.

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Hertig, A. T., and B. Barton 1972 The female reproductive system. In: Handbook of Physiology. E. B. Astwood and R . 0. Greep, eds. Amer. Physiol. Soc., Washington, D.C., pp. 317-348.

Hisaw, F. L. 1947 Development of the Graafian follicle and ovulation. Physiol. Rev., 27: 95-119.

Humphreys, W. J., B. 0. Spurlock and J . S. Johnson 1974 Critical point drying of ethanol infiltrated, cryofractured biological specimens for scanning electron microscopy. In: Scanning Electron Microscopy, 1974.0. Johari and I. Corvin, eds. Proc. Seventh Annual Scanning Electron Microsc. Symp., ITT Research Institute, Chicago, Illinois, pp. 275-282.

Kang, Y. H. 1974 Development of the zona pellucida in the r a t oocyte. Am. J . Anat., 139: 535-566.

Lipner, H. 1973 Mechanism of mammalian ovulation. In: Handbook of Physiology, Vol. 11. Female Reproductive System, Part 1. Section 7: Endocrinology. R. 0. Greep, E.


B. Astwood, and S. R. Geiger, eds. Amer. Physiol. Soc., Washington, D.C., pp. 409-437.

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1970 The role of the zona pellucida in the development of mouse eggs in uiuo. J. Embryol. Exp. Morph., 23: 539-547.

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Peters, H., A. G. Byskov, R. Himelstein, A. Braw and M. Faber 1975 Follicular growth: The basic event in the mouse and human ovary. Reprod. Fertil., 45: 559-566.

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Modlinski, J. A.

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Schwartz, Neena B. 1974

PLATE I

EXPLANATION OF FIGURE

1 Survey scanning electron micrograph of a 28 day old mouse ovary in which two complementary halves of the dissected ovary are seen. Numerous follicles with oocytes can be discerned in the cortex of both halves. Ox, oocyte which has been slightly “disconnected’ from granulosa cells; OV, ovarian epithelium. X 160.

346

OVARIAN FOLLICLES OF MAMMALS E Anderson. R F Wilkinson. G Lee and S Meller

PLATE 1

PLATE 2

EXPLANATION OF FIGURES

2 A small portion of a follicle showing granulosa cells (G), vascularized (V) theca interna (TI) and nonvascularized theca externa (TE). X 700.

Section through a follicle that illustrates some of its anatomical and physiological compartments. 0, oocyte; ZP, zona pellucida; BM, basal lamina or membrana limitans; G, granulosa cells; CE, Call-Exner body. X 500.

4 Section through a Call-Exner body (CE). G, a portion of a granulosa cell resting on the limits of the fibrillar component of a Call-Exner body. X 30,000.

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The inset in this plate is a photomicrograph of a mouse oocyte encompassed by a single layer of granulosa cells. x 100.

5 Scanning electron micrograph of a preantral follicle from a 2 day old mouse ovary. Unidentified small spherical bodies can be seen in the oocyte (arrows). Note that the granulosa cells (G) are generally cuboidal. X 2,700.

Higher magnification scanning electron micrograph of the memhrana limitans of an 18 day old mouse follicle. The collagen fibers can be seen where the theca interna has pulled away from the granulosa cells. X 5,800.

7 The tissue illustrated in this transmission electron micrograph was prepared in tannic-acid-containing fixative. The procedure enhances structures rich in plysac- charides. Notice the fibrous lamina a t the basal aspect of the granulosa cells (arrow), subtended by numerous electron-opaque collagen-like fibers. X 36,000.

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8 Scanning electron micrograph of the outer surface of the zona pellucida from a 21 day old mouse ovarian follicle. The larger depressions (large arrow) are interpreted as sites where pseudopod-like granulosa cell processes associate with the zona. The smaller fenestrations (small arrow) are probably “tracks” along which microvillar extensions of granulosa cells traverse the zona. X 19,000.

Transmission electron micrograph of the oocyte-zona pellucida-granulosa cell relationship. In this preparation, from a 6 day mouse ovarian follicle, one sees granulosa cell pseudopodia and electron-opaque microvilli as well as the more electron-lucent oocyte microvilli that traverse the zona. Cell processes from granulosa cells account for openings illustrated in figure 8. X 20,000.

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10 Scanning micrograph of a rabbit antral follicle that had been prepared without thiosemicarbazide treatment. Several blood vessels of the theca interna are marked at the arrows. x 170.

Higher magnification of a region of the preovulatory follicle seen in figure 10. Here features of endothelial cells (arrow), blood vessels and erythrocytes (arrow) illus- trate the value of this method to study the blood vasculature of the follicle. X 3,400.

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EXPLANATION OF FIGURE

12 Scanning electron micrograph of granulosa cells, zona pellucida, and oocyte from an early antral (28 day) ovarian follicle. The disk-like structures a t the arrows are interpreted to be isomorphic with gap junctions. X 4,800. Inset A is a transmission electron micrograph showing a gap junction between two granulosa cells from lanthanum impregnated material. Inset B is a freeze-fracture replica of the P-face of a gap junction between two granulosa cells. Inset A, X 50,000; inset B, X 40,000.

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13 Scanning electron micrograph of the early antrum (A) in a rabbit ovarian follicle. Note the relatively smooth-walled appearance of the cavity. Also shown is an erythrocyte, probably from the vasculature of the theca interna. X 1,400.

Scanning electron micrograph of a preovulatory rabbit follicle. Smooth-surfaced bolus filling antral cavity is coagulated antral fluid. Cellular elements embedded in the matrix of the antral fluid are marked a t the arrows. These may he cells of the cumulus oophorys. X 140.

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EXPLANATION O F FIGURES

15 Scanning electron micrograph of the surface of a large preovulatory rabbit follicle in an ovary removed 11 hr post-coitus. Regions shown in this micrograph are (1) apical, (21 intermediate, and (3) basal. X 80.

Scanning electron micrograph of a small portion of the apical region of the ovarian follicle illustrated in figure 15. Note the flattened ovarian epithelial (OE) cells, some of which are separated from each other by large spaces; some have been exfoliated, revealing the fibers of the tunica albugenia (TA). X 250.

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17 A scanning electron micrograph of the apical surface of the preovulatory follicle. The flattened epithelial cells contain few microvilli and are separated from each other by relatively large spaces. X 1,400.

A scanning electron micrograph of cells from the intermediate zone. The cells are dome-shaped with relatively few microvilli on their apical surfaces compared with the many on their lateral surfaces. X 1,200.

19 A scanning electron micrograph from the basal zone. Note that the cells are covered with many microvilli. X 800.

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20 A scanning electron micrograph of the ovarian surface from a rabbit 11 hr post- coitus. The large circular area has been identified as a papilloma (P). X 700.

21 A high magnification scanning electron microscope image of a region of the papilloma in figure 20, depicting some of its cellular population. X 2,000.

A semi-thin section, toluidine blue stained, of the papilloma illustrated as figure 20 that had been used for prior SEM. V, vascular elements; GP, gold-palladium coating. X 800.

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EXPLAiVATfON O F FIGURE

23 A photomicrograph of a semi-thin section of a toluidine blue stained ovulated folli cle. 0, oocyte; CO, cumulus oophorus; CAF, coagulated antral fluid. X 700.

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Documents

A correlative microscopical analysis of differentiating ovarian follicles of mammals