Theca Cells May Be Present at the Outset of Follicular Growth1

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1 BIOLOGY OF REPRODUCTION 44, (1991) Theca Cells May Be Present at the Outset of Follicular Growth1 ANNE N. HIRSHFIELD Department of Anatomy, University of Maryland, School of Medicine, Baltimore, Maryland ABSTRACT Opinions differ concerning when theca cells arise during follicular growth. Theca cells cannot be distinguished from ovarian stroma cells on the basis of morphology alone, However, long.term continuous infusion of ]thymidine (5H-TdR) thorougily labels theca cells of large and medium-sized follicles whereas most stromal cells remarn unlabeled. The objective of this study was to determine when, during the course of follicular development, labeled, squamous cells located just outside the follicular basement membrane (putative theca cells) first become apparent, Rats were given infusions of 3H-TdR for various periods of time. Autoradiographs were prepared from 2-prn.thick plastic sections of ovaries. Morphologically recognizable theca cells of large and medium-sized follicles were sharply delineated from surrounding stroma cells by their labeling pattern. Mthougis un ilaminar follicles lacked morphologically distinct theca layers, they all were closely enveloped by labeled cells just outside the basement membrane. Follicles with as few as 4-5 granulosa cells in cross section often had labeled cells on the convex surface of their basement membranes, These observations suggest the possibility that theca cells may already be present at the very outset of follicular growth. INTRODUCTION The walls of a mature preovulatory follicle contain three distinct tissue types: membrana granulosa, theca intema, and theca externa. Primordial follicles, however, appear to contain only granulosa cells; morphologically distinct theca layers are lacking [1]. Investigators disagree on the time when theca cells arise during the course of follicular development. This issue is of central importance for understanding ovarian function and must be resolved before factors directing theca cell differentiation can be identified. Empirical data bearing upon the problem consist exclusively of morphological evidence. In histological preparations, granulosa cells are readily distinguished at the light microscope level from other cells in the surrounding ovarian tissue because they are enclosed within a delimiting basement membrane. No such delimiting feature encloses theca cells, and they are thus difficult to distinguish from other extra-follicular ovarian cells on the basis of morphology alone. However, when rats are given continuous infusions of [3H]thymidine (3H-TdR) [2], uptake of 3H-TdR by theca cells of medium and large follicles readily distinguishes them from stromal ovarian cells which usually are much less mitotically active. Theoretically, long-term continuous infusion of 3H-TdR could be used to identify theca cells in smaller follicles as well. The objective of this study was to determine when, during the course of follicular development, labeled cells just outside the follicular basement membrane (putative theca cells) first become apparent. Accepted February 4, Received November 20, This work was supported by NSF Grant DCB Animals MATERIALS AND METHODS Sprague-Dawley-derived rats of various ages were obtained from Zivic-Miller Laboratories (Alison Park, PA) and housed in a temperature- and light-controlled room with a 12L: 12D light cycle (lights-on at 0600 h). They were maintained under the supervision of a licensed veterinarian in accordance with the principles set forth in the NIH guide for the care and use of laboratory animals. Continuous 3H-TdR Infusion Rats were given an infusion of 3H-TdR to label proliferating cells [2]: Aizet Osmotic Minipumps (model 2ML1; Alza Corporation, Palo Alto, CA) were filled with [3H]thymidine (3H-TdR; 20 Ci/mmol, 1 mci/mi in distilled water; New England Nuclear, Boston, MA) and primed in saline for at least 2 h at room temperature. Manufacturing specifications for these pumps indicated that they were designed to deliver 8.64 p.1/h for up to 7 days. Rats were lightly anesthetized with ether and a pump was inserted under the skin of the back. Rats were killed at various times after the pumps were inserted. Table 1 lists the ages of the rats used for this study, their hormone treatments if any, and the duration of 3H-TdR infusions that they were given. Histology and Autoradiograph-y Ovaries were removed, cleaned of adherent fat, and fixed for at least 72 h in Kahle s fixative [3]. Tissues were embedded in glycolmethacrylate (HistoResin, LKB, Piscataway, NJ), then sectioned at 2 p.m. Every tenth section was mounted on a subbed glass slide [4]. Slides were dipped in NIB 1157

2 1158 HIRSHFIELD TABLE 1. Animals used for study of labeled putative theca cells. Age Treatment 3H-TdR (h) n Adult lgdays Odays Lactating Adult days ecg + hcg 72 1 Adult Pregnant days Adult Adult nuclear research emulsion (Kodak, Rochester, NY), and exposed in the dark at 4#{176}C. Test slides were developed at monthly intervals to monitor exposure. Autoradiographs were developed when there were silver grains over the nucleus of each labeled granulosa cell (approximately 6 mo exposure time). Slides were developed with D19 developer diluted 1:1 with distilled water (for 4 mm), fixed in Kodak fixer (for 5 mm) with all solutions at room temperature, then stained with Lee s methylene blue-basic fuchsin stain [5]. Analysis of Autoradiographs Random sections from each ovary were scanned under oil at 630x magnification to locate very small follicles (<30 granulosa cells in the cross section). Representative follicles were photographed with a Zeiss photomicroscope. RESULTS Granulosa cells constituted the preponderance of labeled cells in all the ovaries examined. Numerous labeled cells were scattered throughout the extrafollicular compartment (region outside the follicular basement membrane) as well. Some of these labeled cells could be identified as endothelial cells because they lined vascular channels. Other cells, presumed to be fibroblasts, were characterized by large rounded nuclei and were scattered individually among unlabeled parenchymal cells, particularly in corpora lutea and in elements of secondary interstitial tissue. The prevalence of labeled extrafollicular cells varied markedly between individuals. Large antral follicles contained clearly recognizable theca layers; the cells in these regions were heavily labeled in ovaries of all rats given long-term continuous infusions of 3H-TdR In contrast, the stromal tissue surrounding these large follicles contained few labeled cells. The transition from the labeled to the unlabeled regions sharply delineated the limits of the theca layer of the follicle (Fig. 1), which would have been difficult to discern by morphological criteria alone. Morphologically recognizable theca cells of smaller follicles (multilayered, preantral follicles) were also heavily labeled, particularly in ovaries of rats that had been given 3H-TdR infusions lasting longer than 48 h (Fig. 2). Unilaminar follicles lacked morphologically recognizable theca cells. However, labeled cells were very often found in the location where theca cells would have been expected to be: closely applied to the convex surface of the follicular basement membrane (Fig. 3). Even the very smallest follicles, containing no more than 4 or 5 squamous granulosa cells in cross section, were frequently found with labeled cells at the convex surface of their basement membranes. The number of labeled cells along the outside of the follicle increased with increasing follicular size. DISCUSSION Studies of other organs have led to the hypothesis that progenitors of all the component cell types that make up complex tissues are assembled together at the very outset of the growth process [6]. However, widely accepted theories of folliculogenesis imply that the ovary has a different timetable of cell assembly and tissue growth. Unlike other tissues, an essential element of the follicle, the theca cell, is believed to be lacking at the outset of follicular growth and is not thought to arise until much later in development [1,7,8]. Direct experimental evidence regarding the ontogeny of theca cells is lacking. Extant literature is confined, for the most part, to speculations based on conventional histological studies [6-13] because the identification of young granulosa and theca cells must be based exclusively on their location in histological preparations with relation to other ovarian structures. Granulosa and theca cells lack distinctive functional characteristics until the penultimate stages of follicular growth. Granulosa cells are easy to distinguish from other ovarian cells in histological preparations because they are enclosed within a basement membrane. In contrast, theca cells are not readily distinguishable from nearby stromal tissue; they are not contained within any delimiting structures such as a basement membrane, and they cannot be distinguished from fibroblasts and other connective tissue cells by morphological criteria alone. Thus, theca cells may be present in primordial or small primary follicles, although they may not be recognizable in histological sections. In this paper, I have reported that continuous infusion of 3H-TdR for extended periods of time revealed labeled cells located just outside the basement membranes of very small follicles. These proliferating cells may be nascent theca cells, because, like theca cells of larger follicles, they are located on the convex surface of the follicular basement membrane. If these cells are indeed young theca cells, then all components of the follicle may be present at the very outset of follicular growth, suggesting that the ovary may have greater similarities to other tissues than originally thought.

3 ONTOGENY OF THECA CELLS 1159 FIG. 1. Long-term continuous infusion of 3H-TdR labeled nearly all theca cells of large healthy follicles whereas most interstitial cells remained unlabeled. The figure shows part of the follicular wall of a large antral follicle; a small portion of the antral cavity is visible at far left. Small arrows indicate the follicular basement membrane. The cells to the left of the basement membrane are granulosa cells. Large arrows delineate the outer limits of the theca layer. The unlabeled cells to the right of the theca layer are stromal cells. An element of the secondary interstitial tissue can be seen in the upper right corner of this frame. (Adult rat, 168-h labeling period, x630j These observations were unexpected in the light of longstanding and widely accepted hypotheses of theca cell ontogeny. In 1947, Hisaw [9] stated that primordial follicles at first do not have a theca interna... When the oocyte begins to grow, a theca interna is formed from the contiguous connective tissue. In 1948, Dubreuil [10] maintained that it is easy to verify that... the theca gland... always first appears in general, at the moment when the multiplying granulosa cells begin to secrete follicular fluid. He postulated that a substance emanating from granulosa cells of medium-sized follicles induced surrounding stromal cells to differentiate into theca. Dubreuil s theory has been reiterated by others [11, 12] and was recently elaborated upon in a review by Erickson and colleagues [13]:... The theca interna first appears during the secondary stage when the oocyte is fully grown and the follicle has acquired 2 to 3 layers of granulosa cells. At this time a signal is generated (most likely by the follicle itself) which causes a stream of cells to migrate to the outer surface of the follicle... This newly acquired connective tissue layer provides the forerunners of both the theca interna and the theca externa cells. Experimental evidence to support, or refute, these hypotheses is lacking. When an enzymatic method was used to dissociate follicles from hamster ovaries, follicles with less than 7 ( stage 6 or less) layers of granulosa cells lacked evidence of a theca layer whereas follicles >7 layers of granulosa cells ( stage 7 ) had prominent theca layers [14]. This was interpreted to suggest the possibility of definitive onset of thecal cell formation between stages 6 and 7. However, an alternative explanation for these observations is that the properties of the extracellular matrix between theca cells changes as the follicles mature, becoming less susceptible to collagenase degradation by stage 7 of growth. My own histological preparations of intact hamster ovaries revealed morphologically distinct theca layers in much smaller follicles (at least as early as 3-4 layers of granulosa cells). My observations of rat ovaries reported here are also open to multiple interpretations. One alternative explanation is that the labeled theca cells that I observed sur-

4 1160 HIRSHFIELD FIG. 2. Labeling patterns following continuous infusion of 3H-TdR delineated theca cells of preantral follicles from the surrounding stromal tissue. (A) Preantral follicle with about 64 granulosa cells in the cross section, ovary of a pregnant rat (Day 12 of pregnancy), 168-h continuous infusion, x630. (B) Follicle with about 30 granulosa cells in largest cross section, adult rat, 48-h infusion, x400. (C) Follicle with 18 granulosa cells, adult rat, 168-h infusion, x630. Some putative theca cells are indicated by arrows. (D) Follicle with 14 granulosa cells, 19-day-old rat, 24-h infusion, x630. Some putative theca cells are indicated by arrows. The regions of intense labeling seen at the top of A and at the bottom of C are portions of theca layers of much larger follicles. rounding very small follicles are merely fibroblasts or other connective tissue cells that happen to be proliferating in the vicinity of follicles that have just begun to grow. If the labeled cells seen just outside the follicular basement membrane were simply stromal/connecuve tissue cells that chanced to be in that particular location, one would expect to encounter more follicles associated with such labeled cells in ovaries that had a great deal of label in the extrafollicular compartment, and fewer follicles in association with such labeled cells in ovaries that had scant label in the extrafollicular compartment. However qualitative evaluations suggested that there was no correlation between the frequency of labeled cells closely applied to the external surface of unilaminar follicles and the density of labeled stromal cells in other compartments of the ovary. Ovaries that were nearly devoid of label in the interstitial compartment frequently contained many unilaminar follicles with labeled putative theca cells. FIG. 3. Very small follicles also had labeled cells on the convex surface of the follicular basement membrane. These could be theca cells and are indicated by arrows. (A) Follicle with 10 granulosa cells, adult rat, 96- h infusion, x630. (B) Ten granulosa cells, adult rat, 96-h infusion, x630. (C) Nine granulosa cells, adult rat, 336-h infusion, x504. ID) Eight granulosa cells, adult rat, 168-h infusion, x504. (El Seven granulosa cells, adult rat, 168-h infusion, x504. (F) Nine granulosa cells, adult rat, 24-h infusion, x400. (G) Five granulosa cells (none are labeled), 20-day-old rat, 48-h infusion, x630. (H) Five granulosa cells (1 is labeled), 19-day-old rat, 24-h infusion, x630, (I) Five granulosa cells (none are labeled), adult rat, 96-h infusion, x504.

5 ONTOGENY OF THECA CELLS 1161 IL. -,

6 1162 HIRSHFLEW The hypothesis that theca cells may be present at the very outset of follicular growth suggests new avenues to explore when seeking answers to questions concerning the regulation of early developmental processes such as the onset of follicular growth. Several recent studies suggest that theca cells produce growth factors which influence granulosa cell proliferation [15, 16]. Although these studies focused exclusively on granulosa and theca of large follicles, it would be logical, by extension, to postulate that growth of cells in smaller follicles may also be regulated by growth factors emanating from theca cells. Because they are enclosed within a basement membrane, granulosa cells are considered to be epithelioid cells. Theca cells, located outside the basement membrane, are usually considered to be derived from and to remain within the mesenchymal compartment [12, 17, 18]. In other tissues, mesenchymal cells have a major influence on the growth and differentiation of epithelial cells [19]. The observations reported here suggest that epithelial/mesenchymal interactions between granulosa and theca cells may play a role in regulating cell growth and differentiation not only in the final stages of follicular development but also in the earliest stages of folliculogenesis. As long as specific cellular markers for immature theca cells remain unavailable, debates concerning thecal cell ontogeny will remain unresolved. It is not possible at this time to conclusively prove that theca cells are either present or absent at the outset of follicular growth. Until this issue is resolved by clear-cut empirical evidence, the possibility that theca cells may already be present in very small follicles should not be dismissed out of hand. ACKNOWLEDGMENT Jannin Simons prepared the autoradiographs for this project. REFERENCES 1. Mossman HW, Duke KL. Comparative Morphology of the Mammalian Ovaiy. Madison, WI:University of Wisconsin Press; 1973: Hirshuield AN. Continuous [3H]thymidine infusion: a method for the study of follicular dynamics. Biol Reprod 1984; 30: Gurr E. A Practical Manual of Medical and Biological Staining Techniques. Londonleonard Hill Books Ltd; Rajaniemi I-lJ, Midgley AR. Autoradiographic techniques for localizing peptide hormones in target tissue. Meth Enzymol 1975; 37: Bennett HS, Wyrick AD, Lee SW, NcNeil JH. Science and art in preparing tissues embedded in plastic for light microscopy with special reference to glycol methacrylate glass knives and simple stains. Stain Technol 1976; 51: Zajicek G. Proliferon: the functional unit of rapidly proliferating organs. Med Hypoth 1979; 5: Young WC. The mammalian ovary. In: Young WC (ed), Sex and Internal Secretions, vol 1, 3rd ed. Baltimore:Williams & Wilkins; 1961: Dvorak M, TesarikJ. Ultrastructure of human ovarian follicles. In: Mona PM, Hafez ESE (eds.), Biology of the Ovary. The Hague:Martinus Nijhoff; 1980: Hisaw FL. Development of the Graafian follicle and ovulation. Physiol Rev 1947; 27: Dubreuil G. Sur lexistence dune substance inductrice a action limitee et locale pour Ia metaplasie theca(e des cellules du stroma cortical ovarien. Ann Endocrinol 1948; 9: ByskovAG. sexual differentiation of the mammalian ovary. In: Mona PM, Hafez E5E (eds.), Biology of the Ovary. The Hague:Martinus Nijhoff; 1980: Gurava S5. Biology of Ovarian Follicles. New York:Springer.Verlag; 1985: Erickson GF, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 1985; 6: Roy SK, Greenwald GS. An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects. Biol Reprod 1985; 32: Bendell B Dorrington J. Rat thecal/interstitial cells secrete a TGF-like factor that promotes growth and differentiation in rat granulosa cells. Endocrinology 1988; 123: Skinner MK, Coffey RJ Jr. Regulation of ovarian cell growth through the local production of TGFa by theca cells. Endocrinology 1988; 123: Hiura M, Fujita H. Electron microscopy of the cytodifferentiation of the theca in the mouse ovary. Arch Histol Jpn 1977; 40: Greenblatt RB, McDonough PG. Endocrine histopathology of the ovary. In: Ingiulla W. Greenblau RB (eds.). Endocrinologic and Morphologic Correlations of the Ovary. Springfield, IL:Charles C. Thomas; 1969: Cunha GR. Mesenchymal-epithelial interactions in the growth and development of the urogenital tract. In: Hirshfield AN (ed), Growth Factors and the Ovary. New York:Plenum Press; 1989: 3-13.