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Regular Article |
a Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523
b AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand
c Department of Pathology, Wellington School of Medicine, Wellington, New Zealand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, developmental biology, follicle, granulosa cells, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the early 1900s, three major sources for pregranulosa (i.e., follicle) cells have been proposed: 1) mesothelial cells (i.e., epithelial cells that form the surface of the developing ovary) [4–7], 2) cells derived from a centrally located blastema [8, 9], and 3) mesonephric cells originating from rete tubules/ovarii [10–12]. In addition, it has been suggested that cells from the centrally located blastema and the intraovarian rete cords may, indeed, be similar structures [13]. Based on results from a comprehensive study of ovine fetal ovaries, Zamboni et al. [1] concluded that mesonephric cells derived from the "giant nephron" not only gave rise to an ovarian blastema that served as a major morphogenetic organizer but were also the source of follicle cells that eventually enveloped oocytes to form primordial follicles. During subsequent studies in sheep, our findings were consistent with those of Zamboni et al. [1], in that we observed a close association between mesonephric-derived "cell streams" and "germ-cell nests" at the time of follicle formation, together with the presence of steroidogenic enzyme mRNA and/or protein in both cell streams and somatic cells immediately adjacent to oocytes [14]. Thus, in mice, cats, mink [11, 12], and sheep [1, 14], the prevailing concept regarding the process of folliculogenesis is that the somatic cell component of ovarian follicles is derived from mesonephric cells and, hence, from the intraovarian rete and/or ovarian blastema.
Although Zamboni et al. [1] provided an excellent account of developmental changes that occur during differentiation of the ovine fetal ovary, no observations were made between Day 73 and Day 120 of gestation, a time period that encompasses a critical window during which ovigerous cords regress and primordial follicles form. In a previous study [14], we examined this critical time period; however, extensive analyses including electron microscopy, use of connective tissue stains, and identification of proliferating cells were not performed. Thus, neither study addressed in detail the formation and disappearance of ovigerous cords during ovarian follicle formation or the role of the surface epithelium in these processes. The purpose of the present study was to determine how ovigerous cords are formed and to establish the sequence of events that lead to the disappearance of ovigerous cords and the subsequent appearance of primordial follicles. Accordingly, the histoarchitecture of the fetal ovaries was evaluated by light and electron microscopy beginning on Day 38 of gestation and at selected time points thereafter, including Days 75, 90, and 100, during which definitive primordial follicles form. In an effort to gain new information regarding the origin of follicular cells, pregnant ewes were treated with 5-bromo-2'-deoxyuridine (BrdU), and fetal ovaries were examined to identify proliferating cells at selected stages of development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
To rule out any developmental effects of litter size, female fetuses were recovered at various gestational ages following the transfer of 4- to 5-day-old embryos (three per recipient) as described previously [15]. Fetuses were the result of matings between superovulated Romney ewes (n = 11) and Romney rams (n = 4). A total of 56 female fetuses were recovered from recipient ewes following euthanasia using a barbiturate overdose (Euthatal, 
20 ml i.v. of a solution containing 500 mg/ml; South Island Chemicals, Christchurch, New Zealand).
Light Microscopy
Fetuses were recovered at Days 38, 45, 55, 65, 75, 85, 90, and 100 of gestation. Ovaries were fixed in Bouin fluid and either embedded in paraffin (n = 29 ovaries, each from a separate fetus and a minimum of three per age group) and serially sectioned at a thickness of 5 µm for routine histological evaluation or embedded in methacrylate (Technovit 7100; Kulzer & Co., GmBh, Werheim, Germany) and serially sectioned at a thickness of 30 µm (n = 25 ovaries, each from a separate fetus and a minimum of three per age group). Serial sections were used to reconstruct the arrangement of ovigerous cords within an ovary. The ability to focus at different planes within a 30-µm-thick section permitted the developmental stage of a given follicle to be accurately determined, because the shape (i.e., squamous vs. cuboidal) of the associated granulosal cells could be easily discerned. For routine evaluation of paraffin-embedded tissue, sections were stained with hematoxylin and eosin. To visualize basement membranes, some sections were stained using either PAMS (a microwave-assisted, periodic acid-methenamine silver stain [16]) or a reticular stain [17].
Identification of Proliferating Cells
To monitor location and extent of proliferating cells within fetal ovaries at different stages of development, a total of 21 fetal ovaries (each from a separate fetus and a minimum of three fetuses per age group) were collected on Days 38, 45, 55, 65, 75, and 90 of gestation 1 h after injecting (i.v.) the dams with 250 mg of BrdU (Roche Diagnostics NZ Ltd, Auckland, New Zealand) in 12 ml of normal saline. Fetal ovaries were dissected free of surrounding tissue and fixed at 4°C for 60 min in freshly prepared 4% (w/v) paraformaldehyde in PBS (pH 7.4), embedded in paraffin, and sectioned at a thickness of 5 µm. Cells that had incorporated BrdU were identified using the reagents and procedures supplied with a BrdU-immunohistochemical detection kit purchased from Zymed (San Francisco, CA).
Electron Microscopy
A total of 24 ovaries (each from a separate fetus and a minimum of three per age group) were recovered at Days 38, 45, 55, 65, 75, and 90 of gestation. Ovaries and mesonephroi were dissected free from surrounding tissue and fixed for 18–24 h at 4°C in a solution of 0.1 M cacodylate (pH 7.4) containing 2.5% (w/v) glutaraldehyde and 5% (w/v) sucrose. Tissues were postfixed at room temperature for 90 min in 1% (w/v) osmium tetroxide-0.1 M cacodylate, stained en bloc with 2% (w/v) aqueous uranyl acetate, dehydrated through a graded series of ethanols, and embedded in Polybed 812 (Polysciences, Inc., Warrington, PA). Before selecting areas to be sectioned and stained for electron microscopic examination, 1-µm-thick sections were cut, affixed to glass slides, and stained with toluidine blue. Areas of interest were selected and thin sections prepared. Sections were mounted on 300-mesh copper grids, stained with uranyl acetate and lead citrate, and examined with a Phillips CM 100 transmission electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 38 of gestation, the fetal ovary contained five distinct cell types that could be identified by light (Fig. 1) and electron microscopy (Fig. 2, A–F): 1) mesothelial cells that comprised the ovarian surface epithelium, 2) endothelial cells that formed blood vessels, 3) mesenchymal or stromal cells, 4) pregranulosal or follicular cells, and 5) germ cells or oogonia. The surface epithelium of the ovary consisted primarily of low cuboidal cells. Although these cells were anchored laterally by tight junctions, and by desmosomes at focal points along their basal surfaces, they were loosely arranged and did not rest on a discernible basement membrane (Fig. 2A). In some areas examined, oogonia were occasionally found interspersed among cells that comprised the ovarian surface epithelium (Fig. 2B). Endothelial cells were easily distinguished from surrounding mesenchymal cells by their darker staining cytoplasm, the presence of a lumen, and in some cases, red blood cells within the lumen (Figs. 1 and 2C). Mesenchymal cells were fusiform/stellate in shape and characterized by long, thin cytoplasmic extensions that made contact with surrounding cells (Figs. 1B and 2D). Oogonia were usually spherical in shape, larger than any of the other cell types, and appeared to be more numerous toward the periphery of the ovary (i.e., presumptive cortical region) than in the more central region (i.e., presumptive medullary region) of the developing ovary (Figs. 1A and 2E). Pregranulosal cells were in close association with oogonia (Fig. 3A) and, like oogonia, had an abundance of prominent cytoplasmic organelles. These cells were distinguished from stromal cells, in that they were in direct physical contact with at least one oogonium, were oblong in shape, and had well-developed elements of rough endoplasmic reticulum (Fig. 2, E and F). Pregranulosal cells were characterized by the presence of discontinuous elements (i.e., isolated patches) of basal lamina material on the plasma membrane surface that was in contact with the surrounding extracellular matrix (Fig. 3B). In contrast to mesenchymal and endothelial cells, oogonia and pregranulosal cells often contained lipid droplets (Fig. 2F) and elements of smooth endoplasmic reticulum (not shown) characteristic of steroid-producing cells. As individual ovigerous cords advanced in development, the basal lamina became more prominent and formed a continuous layer isolating the pregranulosal cells and oogonia from the adjacent stroma (Fig. 3C). Following initial contact with germ cells, and concomitant with appearance of the basal lamina, pregranulosal cells developed long, slender cytoplasmic extensions that protruded between oogonia, thus segregating individual oogonia from one another (Fig. 3D).
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Cells that structurally resembled oogonia located in the presumptive cortical region were present in the central, presumptive medullary region of the ovary. However, in contrast to germ cells in the cortical region that were incorporated into ovigerous cords, these cells were distributed sparsely and were almost always found in close association with the walls of blood vessels (Fig. 1B). Although clusters/aggregates of three to five of these "germ cell-like" cells were observed, no indication of direct physical association with adjacent somatic cells was observed, nor were the clusters enveloped in a delimiting basal lamina. Because of their structural similarities to steroidogenic cells and oogonia, these cells have been referred to as interstitial cells in the human [18] and as "lost germ cells" in sheep [19]. To avoid any confusion of these cells with germ cells lost as a consequence of apoptosis, we will hereafter use the term "medullary germ cells."
Formation of Ovigerous Cords (Days 38–75)
The establishment of a pregranulosa cell-oogonium complex marked the initial contact between somatic cells and germ cells and was the first recognizable step in the process of ovigerous cord formation (Figs. 1A and 2E). The plasma membranes of oogonia and pregranulosal cells were in close apposition to one another, and along their surfaces, focal points of physical attachment (i.e., desmosomes) were apparent (Fig. 3A).
Once a pregranulosa cell had made contact with an oogonium, it appeared that these somatic cell-germ cell complexes progressively fused to give rise to tube-like structures (i.e., ovigerous cords) that contained oogonia (Fig. 4). Although the outer wall of ovigerous cords consisted solely of pregranulosa (i.e., somatic) cells that secreted a basal lamina, pregranulosal cells were observed within the central portions of some cords (Fig. 4D). The process of ovigerous cord formation was evident at Day 38 and continued through Day 75 (Fig. 5). All oogonia (except for medullary germ cells) were either associated with pregranulosal cells (Fig. 4, B and C) and in ovigerous cords (Fig. 4, D and E, and Fig. 5) or were dispersed among loosely arranged cells that formed the ovarian surface epithelium (Fig. 4, B and F, and Fig. 5). Similar to seminiferous cords in fetal testes, ovigerous cords were tubular in appearance, avascular (Fig. 4, D–F), and segregated from the stromal compartment by a prominent and continuous basal lamina (Figs. 3C and 6A). However, in contrast to seminiferous cords, no apparent physical barrier (e.g., basement membrane) separating the loosely arranged surface epithelial cells from oogonia and oocytes in ovigerous cords was observed. As a result, and up until Day 75, ovigerous cords appeared to be open-ended near the surface epithelium, thus permitting contact between surface epithelial cells and oogonia not yet incorporated into ovigerous cords (Fig. 4, B, E, and F, and Fig. 6A). Beginning at Day 75 and continuing through Day 100, the surface epithelium gradually became segregated from ovigerous cords by a basement membrane (Fig. 6). As development proceeded, morphological differences among various cell types, both outside and within the ovigerous cords, became more pronounced (Fig. 4, D and E, and Fig. 6A), and from Day 38 to Day 75, the ovigerous cords progressively became more extensive, elongated, and convoluted. As a result, the ovarian cortex enlarged and became more clearly defined (Fig. 5).
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Oogenesis, Cell Proliferation, Meiosis, and Cell Death (Days 38–100)
Incorporation of BrdU into proliferating cells was examined on Gestational Days 38, 45, 65, 75, 90, and 100, which encompassed the time frame during which primordial follicles were formed. Before Day 90, proliferating cells were more numerous in the cortex than in the medulla (Fig. 7, A–D). This was due largely to the high proliferative activity of ovarian surface epithelial cells and oogonia. On Day 38 and Day 45, oogonia that had incorporated BrdU were distributed throughout the cortex (Fig. 7B), but on Day 75, labeled cells were more prevalent toward the outer periphery of the cortex (Fig. 7C). Within the stroma of the cortex and medulla, most of the proliferating cells identified, either by incorporation of BrdU or by the presence of mitotic figures using conventional microscopy, appeared to be endothelial cells (data not shown). In contrast to the high proliferative activity of surface epithelial cells and oogonia and, to a lesser extent, endothelial cells (Fig. 7, A–C), the stromal (i.e., mesenchymal) cells and pregranulosal cells within the ovigerous cords were quiescent during the same time periods examined (Fig. 7, E and F). During the interval from Day 55 through Day 90, and at the same time surface epithelial cells and oogonia were proliferating, two other major events were ongoing within ovigerous cords: initiation of meiosis, and apoptosis of germ cells. Germ cells in prophase of meiosis I were first observed on Day 55 and were increasingly prevalent by Day 75. Whereas oocytes in prophase of meiosis I were predominantly located in regions of ovigerous cords closest to the medulla, proliferating oogonia were more prevalent in regions adjacent to the surface epithelium. At the same time that some germ cells were proliferating or initiating meiosis, others were undergoing apoptosis. Based on the location, size, and shape of degenerating cells, most were judged to be germ cells located within the ovigerous cords (Fig. 8). Often, germ cells in the early stages of meiosis were adjacent to germ cells undergoing apoptosis (Fig. 8B). Although large numbers of germ cells were undergoing apoptosis, no convincing evidence was obtained to indicate a similar loss of pregranulosal cells within the ovigerous cords (Fig. 8). On Day 100, consistent with the dissolution or regression of the ovigerous cords and the formation of primordial follicles, the chromatin pattern in nuclei of oocytes in primordial follicles was characteristic of the dictyate stage of meiosis (data not shown).
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Emergence of Primordial Follicles from Ovigerous Cords (Days 75–100)
The structural integrity of ovigerous cords was maintained until Day 75, at which time primordial follicles first began to emerge from regions of ovigerous cords located nearest the interface of the cortex and medulla (Figs. 5 and 9). Between Days 75 and 90, primordial follicles appeared to form without oocyte-pregranulosa cell complexes ever being directly exposed to somatic cells outside the ovigerous cord structure (Fig. 9, A–G). Based on light and electron microscopic observations, the basal lamina of forming follicles first appeared to be laid down within intact cords in such a manner that oocyte-pregranulosa cell complexes separated from one another, either singly or in small groups (Fig. 9, D–F). Individual follicular units or primordial follicles emerged completely enveloped within a continuous basal lamina (Fig. 9G). However, it is important to note that until the time of "follicle breakout," the basal lamina was restricted to the outer wall of the ovigerous cords. Thus, although cytoplasmic projections extending from pregranulosal cells that formed the wall of ovigerous cords, as well as projections from pregranulosal cells situated within the central portions of the cords, segregated the oocytes from one another, no basal lamina was involved.
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Primordial follicles that emerged from ovigerous cords consisted of either an oocyte surrounded by a single layer of flattened (i.e., squamous) granulosal cells (i.e., type 1) or an oocyte surrounded by a single layer of granulosal cells, except that one or more of the granulosal cells were cuboidal in shape (i.e., type 1a) (Fig. 9G). To exclude the possibility that classification of granulosal cells as cuboidal was an artifact simply due to the plane of the section examined, cell shape was verified using 30-µm-thick plastic sections.
In some areas, two or more follicular complexes appeared to be enveloped within a common basal lamina (Fig. 9E). In some of these complexes, small amounts of basal lamina material between the complexes was observed, indicating that an additional process was taking place to separate individual follicular units (Fig. 9, D–F). Sometimes, as oocyte-pregranulosa complexes were isolated from ovigerous cords, irregular-shaped follicles were formed that were characterized by an uneven distribution of pregranulosal cells around a single oocyte. An extreme example included a "comet-like" arrangement of pregranulosal cells around the oocyte (data not shown). On rare occasions, more than one oocyte shared a single layer of granulosal cells and common basal lamina (data not shown). A conceptual model of potential ways by which normal and abnormal follicles might form from ovigerous cords is illustrated in Figure 10.
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The gradual dissolution of the ovigerous cords as a result of follicle formation proceeded from the interface of the cortex and medulla toward the periphery of the cortex. The sequential and progressive changes were such that, on Day 90, the cortex of the fetal ovary was subdivided into two distinct zones (Fig. 11); one that contained germ cells with the remaining intact portions of ovigerous cords, and one that contained type 1 and type 1a primordial follicles. By Day 100, ovigerous cords (zone 1) had essentially disappeared, and the cortex contained type 1, type 1a, and also type 2 follicles (Fig. 11). The distribution of these follicles was such that the type 2 follicles (i.e., primary follicles with one, but less than two, complete layers of cuboidal granulosal cells) were located at the interface of the cortex and medulla. Hence, the more developmentally advanced, growing type 2 follicles were closer to the medulla than the population of nongrowing follicles (i.e., type 1 and type 1a follicles) (Fig. 11).
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From Day 75 through Day 100, when follicle formation occurred and the ovigerous cords were regressing, the cords became increasingly isolated from the surface epithelium by the progressive development of an intervening basement membrane that separated them (Fig. 6, B–F). On Day 90, the ovarian surface epithelium was more organized compared to that during earlier stages of development (Day 38, Fig. 1A; Day 55, Fig. 4B; Day 90, Fig. 4F and Fig. 6, E and F). By Day 100, epithelial cells were no longer loosely arranged, rested on a distinct basement membrane, and were separated from the newly formed follicles (Fig. 11).
Medullary Germ Cells
As described above, cells that structurally resembled oogonia were present in the ovarian medulla from Days 38 to 100 (Fig. 12). Although much fewer in number (<10%) than germ cells in ovigerous cords, these medullary germ cells contained lipid droplets, elements of smooth endoplasmic reticulum, and an extensive Golgi apparatus. In addition, some had incorporated BrdU, and some had entered prophase of meiosis I (Fig. 12, insert). Although some medullary germ cells remained in clusters of three to five cells, most were distributed as isolated single cells. From Day 75 to Day 100, many of the medullary germ cells were located within the rete tubule network in the medullary region of the developing ovary. Although no evidence was found at any of the fetal ages examined that medullary germ cells developed into follicles, their fate was not followed beyond Day 100.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of this study do not provide insight regarding the origin of pregranulosal cells present within the fetal ovary at Day 38 of gestation. At this time, most of the oogonia are in direct physical contact with one pregranulosa cell (Figs. 1 and 2); thus, the number of pregranulosal cells can be estimated to be approximately 30 000 [15]. It seems likely that the precursors of these first pregranulosal cells originate from the mesonephros, as proposed by Zamboni et al. [1]. The initial contacts between oogonia and pregranulosal cells are characterized largely by adherens-like junctions including desmosomes. At Day 38, the oogonia-pregranulosa cell complexes were more numerous toward the periphery of the ovary. Both before and after Day 38, germ cells express the cell surface receptor c-kit mRNA/protein. By contrast, from Day 24 to Day 90, the ligand for c-kit, namely stem cell factor (SCF), is initially expressed (i.e., mRNA/protein) throughout the ovary, but after Day 35, it becomes increasingly restricted to pregranulosal cells and the surface epithelium [21]. It is, therefore, reasonable to speculate that the preferential location of oogonia toward the surface epithelium is based on a "chemotactic" attraction of one cell type to the other, and that the c-kit-SCF interaction represents one of these.
The proliferation of oogonia occurs despite the close physical association of this cell with a pregranulosa cell. Moreover, the associations of pregranulosal cells with oogonia at Day 38 preceded ovigerous cord formation. From Day 38 onward, ovigerous cords appeared to arise from a progressive fusion of pregranulosa cell-oogonium complexes and with the arrangement of these cellular components such that pregranulosal cells formed the outer wall of the ovigerous cord. At Day 45, ovigerous cords were well delineated by a basal lamina and appeared to completely enclose the pregranulosa cell-oogonium complexes within the cortical regions of the ovary. Despite tracing numerous ovigerous cords, often with membrane-specific stains, no evidence was found for open connections or continuity between the cords and the ovarian stroma within either the cortical or the cortical-medullary regions. However, very clear evidence was found (Figs. 1, 4, and 6) that the cords were open to the surface epithelium at least until Day 90. Thus, we conclude that, after Day 38, dividing oogonia can recruit a somatic cell partner only from the ovarian surface epithelium. Evidence in support of this notion can be obtained from the BrdU results. Within the cords, the major population of dividing cells was oogonia, mainly near the ovarian surface epithelium; few, if any, somatic cells within the cords were labeled with BrdU. Concomitant with the presence of proliferating oogonia was a highly proliferative surface epithelium together with evidence at both the light and electron microscopic levels for oogonia making contact with surface epithelial cells. Consistent with these findings are those of Tisdall et al. [21], who showed c-kit mRNA/protein expression in oogonia and SCF mRNA/protein in somatic cells within cords and in cells of the surface epithelium at Days 55, 75, and 90.
The maximum number of germ cells present within the fetal ovary of sheep is found at Day 75 (i.e., 805 000) [15]. If it is assumed that each of these germ cells is in contact with at least one somatic cell, then this represents an increase over the number at Day 38 by 775 000 cells. Therefore, most (i.e., >95%) of the pregranulosal cells at Day 75 are of surface epithelial origin. Although it could be argued that this would not be true if the ovigerous cords had open connections to migrating cells from the ovarian stroma or central (i.e., medullary) blastema, no evidence was found for the existence of such connections. Furthermore, newly formed oogonia are located in the outer cortical regions of the ovigerous cords and appear to associate only with somatic cells expressing SCF at the surface epithelium [21]. Thus, collectively these data support the hypothesis that most of the somatic cells (i.e., granulosal cells) associated with germ cells originate from the surface epithelium.
Between Day 75 and Day 90, >75% of germ cells die [15], presumably by apoptosis [22]. The substantial loss of germ cells is not accompanied by any evidence for pyknosis of the pregranulosal cells. This phenomenon of germ cell apoptosis without concomitant loss of somatic cells by apoptosis has been reported for other species (e.g., human [23]). Experimental evidence from mice, both in vivo and in vitro, indicate that the presence of c-kit and its ligand SCF are critically important for the survival of oocytes entering meiosis (see Driancourt et al. [24]). In the sheep, c-kit mRNA expression was either absent or low (i.e., near background levels) in meiotic germ cells within the cords at Day 75 compared to the expression in oogonia at Day 55. The pattern of SCF mRNA at Day 75 indicated intense expression in cells at or near the surface epithelium and in pregranulosal cells near the top of the cords. However, farther within the cord, the intensity of the SCF mRNA signal was lower where germ cells would be expected to undergo meiosis. In contrast to the mRNA, the presence of protein for c-kit and SCF was detectable in oocytes and somatic cells, respectively; however, a declining concentration gradient was evident for both proteins from the outer to the inner cortex [21]. These observations in fetal sheep are consistent with the notions that c-kit and SCF are survival factors and that germ cells entering meiosis are more likely to enter apoptosis than oogonia because they have lower concentrations of c-kit and/or are exposed to lower levels of SCF protein. Within the ovigerous cords, the germ cells are easily recognizable, and from Day 55, an increasing number, especially those not immediately adjacent to the surface epithelium, can be observed to be undergoing pyknosis, meiosis, or to have reached the dictyate stage of meiosis, whereas those closest to the surface epithelium are present as oogonia. Thus, given that most of the pyknotic cells within the ovigerous cords are germ cells, the consequence is that in excess of 600 000 pregranulosal cells are left behind. During fetal life, newly formed primordial follicles may contain between 3 and 136 granulosal cells around the oocyte [14, 25]. The question, therefore, arises regarding how these follicles may form with such a variable number of granulosal cells, because between Days 38 and 90, most germ cells within an ovigerous cord are associated with at least one or, possibly, up to five somatic cells. What is known is that the first follicles form at the base (i.e., that portion of a cord closest to the medulla) of ovigerous cords, and that somatic cells within the cords do not appear to divide. Therefore, it seems reasonable to hypothesize that, as germ cells die, the surviving somatic cells may form associations with the remaining oocytes. In newly formed primordial follicles, a highly significant correlation exists between the diameter of the oocyte and the number of granulosal cells [25]. In the present study, the oocytes within the cords were highly variable in diameter (data not shown). Therefore, the number of somatic (i.e., pregranulosal cells) associating with oocytes within the cords likely is, in part, dependent on the number of surplus cells nearby and on the surface area of the oocyte membrane available for contact. How these contacts are made and the factors responsible are unknown. Examples of how germ cells may acquire additional pregranulosal cells within an ovigerous cord are outlined in Figure 13. Collectively, the evidence from the present study and from others [15, 23, 24] provides support for the hypothesis that germ cell atresia is a means by which each surviving oocyte gains additional pregranulosal cells within an ovigerous cord before follicle formation.
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A variation on the above, but not a mutually exclusive, interpretation is that several oocyte-pregranulosa cell complexes may isolate themselves from ovigerous cords by surrounding themselves with a basement membrane. Results from histological studies indicate that these complexes might be further modified following germ cell pyknosis and subsequent remodeling of the surviving oocyte and somatic cell population to form a primordial follicle (Fig. 13). Another possibility to be considered is that the oocyte-somatic cell complexes might become exposed at the base of the ovigerous cord following regression of the basement membrane, so that the adjacent stromal "cell streams" contribute additional cells before follicle formation [14]. However, no evidence for this was found. Careful examination of newly formed follicles and oocyte-somatic cell complexes at the base of the cords between Day 75 and Day 90 show that the principal process occurring was that granulosal cells and adjacent oocyte(s) within the cords sequentially isolated themselves from the cord and surrounding stroma by forming a basal lamina. Moreover, all the evidence indicated that this process took place without the oocyte-granulosa cell complex being exposed to the stromal cell environment.
The results support the notion that, as discrete primordial follicles emerged, the associated granulosal cells could be present either as a concentric layer of squamous cells giving rise to type 1 primordial follicles or as a mixture of squamous and cuboidal cells giving rise to type 1a primordial follicles [14]. The presence of cuboidal cells was found not to be an artifact of the plane of sectioning. The question regarding what triggers the pregranulosal cells around an oocyte to synthesize basement membrane to isolate a follicle from the ovigerous cord remains unknown. An attractive candidate that is known to be essential for follicle formation is Fig 
. This oocyte-specific transcription factor might be involved in attracting pregranulosal cells to the oocyte and directly or indirectly organizing the pregranulosal cells to synthesize basement membrane [26]. Given the importance of bone morphogenetic proteins (BMPs) in embryonic or fetal development, the role of oocyte- or somatic cell-derived BMPs in follicle formation cannot be ruled out [27].
Collectively, the aforementioned evidence supports the hypothesis that follicle formation takes place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla. At Day 90 to Day 100, the number of primordial follicles corresponds to the maximum number that form in a sheep ovary [20]; on average, this corresponds to approximately 100 000 follicles. Lundy et al. [25] reported that the mean total number of granulosal cells is 16 in a type 1 follicle and 32 in a type 1a follicle. Because the total number of germ cells in a fetal ovary at Day 75 is approximately 850 000 and each germ cell is associated with between one and five somatic cells (i.e., 85–425 x 104 somatic [i.e., pregranulosa] cells), it is possible to argue in favor of hypothesis 3, namely that most of the granulosal cells required at follicle formation are derived solely from the pregranulosa cell population within the ovigerous cords. To verify this claim, further morphometric analyses of somatic cell populations within ovigerous cords will be required.
The results of the present study are consistent with the concept that granulosal cells arise from at least two sources, namely the mesonephros (or blastema) until Day 38 and, thereafter, from the ovarian surface epithelium. The notion of heterogeneity of granulosal cells in primordial follicles has been reported by many authors (for reviews, see Peters [3] and Hirshfield [28]). Whereas heterogeneity with respect to the origin of granulosal cells is probably a common phenomenon, there may well be considerable species differences in the contributions of various cell types to the primordial follicle. In some species (e.g., mouse, hamster), the surface epithelium is thought not to be essential for follicle formation [29], although recent evidence indicates that epithelial cells can migrate into the ovarian cortex and, thus, be a source of granulosal cells [30]. Moreover, analysis of chimerical mouse ovaries has provided evidence that the surface epithelium and the granulosal cells are clonally related [31]. In humans, rhesus monkeys, rabbits, and beagles, the morphological evidence indicates that the surface epithelium contributes to the pregranulosa cell population in newly forming follicles [5, 7, 32, 33]. It remains to be determined whether sheep represent an extreme example with respect to the surface epithelium being the major contributor of granulosal cells. Irrespective of the origins of granulosal cells, what is known for all species is that follicle formation begins from the innermost part of the cortex and then spreads outward toward the surface epithelium. Therefore, to determine the relative contributions of the central blastema and/or intraovarian rete and surface epithelium to the granulosa cell population, it will be important to study in a detailed, sequential manner 1) the time period over which proliferating oogonia are exposed to the aforementioned cells before ovigerous cords are formed, 2) the time period during which the cords remain open at the surface epithelium before the latter is isolated by basement membrane, as reported herein and by Gondos [7], and 3) whether the ovigerous cords are ever open within the ovarian cortex (e.g., around the cortical-medullary boundary). In sheep, as in the rabbit, human, and beagle [5, 6, 18, 19], some germ cells fail either to pair with a pregranulosa cell or to migrate into the outer cortex and appear to become segregated in the medulla. In any study of folliculogenesis, it is important to monitor the fate of these cells and to determine whether they have a role in this complex process.
This study does not address the issue of where the precursor cells for the theca interna originate. Recently, Quirke et al. [34] have demonstrated that the developing fetal ovary is steroidogenically active from Day 30 to Day 75. It was established that the steroidogenically active cells were initially located at the boundary of the ovarian cortex and medulla and, from Day 55 to Day 75, were confined to the cell streams between the ovigerous cords. These cell streams are associated with the blood capillary network (Fig. 4) [14]. Thus, one possibility is that, following the formation of primordial follicles and the regression of ovigerous cords, the newly formed follicles and cell streams develop associations with one another, and in so doing, the follicle acquires additional steroidogenic cells, which eventually become the theca interna.
In summary, the origins of granulosal cells and sequelae involved in the formation of primordial follicles were examined in sheep. The conclusions are 1) that changes in the histoarchitecture of the developing ovary center around the formation (Day 38) and subsequent regression of ovigerous cords and concomitant formation of primordial follicles (Days 75–100), 2) that the major source (i.e., >95%) of granulosal cells is the ovarian surface epithelium (hypothesis 1), and 3) that the sequential events leading to follicle formation take place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla (hypothesis 2). Moreover, a third and novel hypothesis is proposed whereby the loss of germ cells, but not of somatic cells, by apoptosis within ovigerous cords is a means by which each surviving oocyte gains an appropriate number of pregranulosal cells before follicle formation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (99-2389) and the New Zealand Foundation for Research Science and Technology. 
2 Correspondence: Heywood R. Sawyer, Animal Reproduction and Biotechnology Laboratory, Foothills Campus, Colorado State University, Fort Collins, CO 80523. FAX: 970 491 3557; heywood.sawyer{at}colostate.edu
Accepted: November 12, 2001.
Received: August 21, 2001.
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