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Biology of Reproduction 61, 358-366 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Evidence for Ovarian Granulosa Stem Cells: Telomerase Activity and Localization of the Telomerase Ribonucleic Acid Component in Bovine Ovarian Follicles1

Tina C. Lavranosa, J. Michael Mathisc, Sue E. Lathamb, Bill Kalionisb, Jerry W. Shayd, and Raymond J. Rodgers2,a

a Flinders University of South Australia, Departments of Medicine and b Obstetrics-Gynaecology, Bedford Park, South Australia 5042, Australia c Louisiana State University, Medical Center, Department of Cellular Biology and Anatomy, Shreveport, Louisiana 71130 d Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60–100 µm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6–8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G2/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomeric repeats are critical structures that function in the stability and replication of chromosomes. In normal somatic cells, the length of the telomere repeat sequence decreases during every cell division because of the inability of the lagging strand to replicate the very 5' end of a linear DNA molecule (reviewed in [1]). Telomerase is a ribonucleoprotein complex that adds hexameric (TTAGGG) telomeric DNA repeat sequences to the ends of eukaryotic chromosomes [2]. The telomerase enzyme has two components—a protein catalytic component and an RNA component containing the complementary repeat sequence [3,4]. Telomerase is inactive in normal somatic cells, and shortening of the telomere length to less than a critical level is thought to signal cell senescence (reviewed in [5]). In contrast, telomerase activity is expressed in germ cells [6], in the endometrium [7], in stem cells present in bone marrow and blood cells [8, 9], in the liver [10], and in the epidermis [11]. Thus, telomerase activity is present in regenerative tissues with high proliferative need, where telomere loss would deplete the stem cell populations.

Regeneration and cell proliferation occur in the ovary. The mammalian adult ovary contains tens of thousands of inactive primordial follicles. Each primordial follicle contains an oocyte, arrested in meiosis and surrounded by a single layer of flattened epithelial pregranulosa cells. Each day a few of these follicles become active, at which time the oocyte begins to enlarge—not divide—and the surrounding granulosa cells commence dividing. As the follicle enlarges due to the replication of the granulosa cells, a process that may take many months to occur in species like humans and cattle, a fluid-filled antrum forms. The granulosa cells continue to divide, and as follicle maturation is reached just before ovulation, granulosa cell division decreases and the next phase of meiosis of the oocyte resumes. After the ovulation process, division of granulosa cells ceases and these granulosa cells then differentiate into luteal cells of the developing corpus luteum (for review see [12]).

We have previously postulated that during follicular growth, granulosa cells arise from a population of stem cells. In support of this hypothesis we have emphasized the fact that follicular granulosa cells divide uninhibited by contact with each other [13]. We have also shown that a proportion of granulosa cells can divide without the need for anchorage [14–18]. The cells within the adult that divide in such a way are normally either stem cells or transformed cancer cells [19, 20]. In previous studies, it was shown that telomerase activity is present in human ovaries and testes [6, 21], consistent with the concept that germ line cells exhibit stem cell properties. Conflicting reports have emerged regarding the ovary, some indicating that oocytes express telomerase [22, 23] and others indicating its absence [6]. Although fertilized oocytes are totipotent stem cells and express telomerase, mammalian oocytes do not replicate in the adult ovary. Therefore we considered it unlikely that telomerase would be active in these cells. On the basis of on our previous work we predicted that granulosa stem cells or their dividing progeny would therefore express telomerase.

The aim of this study was to investigate the specific pattern of telomerase activity present in the ovary throughout the development of the ovarian follicle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Bovine ovaries were collected at a local abattoir and placed into Earle's balanced-salt solution (Hepes buffered, without calcium and magnesium). Only ovaries from cyclic, nonpregnant females (as determined by manually palpating each uteri for the presence of a fetus) were collected. Once back in the laboratory, the ovaries were placed into one of three groups and further processed after a brief morphological evaluation. Ovaries were either grated to obtain preantral follicles (60–100 µm), or they were sliced and small antral follicles were dissected out (1 mm in diameter) from the slices. Individual medium-sized antral follicles (3 mm in diameter) or larger antral follicles (6–8 mm in diameter) were scraped to obtain granulosa cells. Follicles and granulosa cells were subsequently allocated to either a telomerase assay or cell cycle analysis. A separate group of ovaries (n = 14) was also collected from the abattoir for the in situ hybridization experiments; these ovaries were also from nonpregnant females. They were trimmed of any extra tissue, bisected, and then placed into 4% paraformaldehyde for return to the laboratory, where they were cut into smaller pieces and then fixed further for approximately 24 h. They were then dehydrated and subsequently embedded in paraffin. Paraffin-embedded bovine ovarian tissue sections (5 µm) were cut, and the sections were used for in situ hybridization experiments.

The cell line used in this study (293: transformed human primary embryonal kidney cells) was obtained from the American Type Culture Collection (Rockville, MD) and cultured in 75-cm2 tissue culture flasks in Dulbecco's modified Eagle's medium/F12 (Trace Biosciences Pty Ltd., Melbourne, Victoria, Australia) supplemented with 10% heat-inactivated fetal calf serum (CSL, Melbourne, Victoria, Australia) and antibiotics (100 µg penicillin/ml, 100 µg streptomycin/ml, 0.25 µg fungizone/ml; CSL). Cells were harvested by treatment with 0.05% trypsin:0.02% EDTA in Earle's balanced-salt solution and then washed with PBS; cell pellets were placed at-70°C until required for assay.

Preantral follicles (60–100 µm) Ovaries were washed with sterile saline and trimmed of any extra tissue; they were then washed twice with M199 medium supplemented with 4.2 mM NaHCO3, 5% fetal calf serum (CSL), and 50 µg/ml gentamycin (Sigma Chemical Co., St. Louis, MO). Under sterile conditions each ovary was then grated on a very fine grater to a depth of approximately 2 mm below the surface of the ovary in 10 ml of medium. Both 500 U/ml collagenase (Worthington Biochemical Corporation, Freehold, NJ) and 100 µg/ml DNase (DN-25; Sigma) were added to a total volume of 20 ml of grated ovarian tissue in M199 medium (supplemented as described above). The tissue suspension was placed in a sealed conical flask and incubated at 37°C in a shaking water bath for 2 h. Follicles within the correct size range (60–100 µm) were then aspirated from the suspension under an inverted microscope and washed twice in sterile PBS (10 mM phosphate, pH 7.4). A total of 120 follicles from each ovary were pooled for each assay (in approximately 100 µl of PBS) and stored at-70°C until assayed, or fixed for flow cytometric analyses.

Small antral follicles (1 mm) Small antral follicles were obtained from ovaries that were washed with sterile saline, trimmed of all extra tissue, and bisected sagittally. Each half of the ovary was placed on a tissue slicer and sliced (approximately 1 mm thick) down to approximately 4 mm from the surface of the ovary. Each ovary slice was placed on a clean glass microscope slide and viewed under a dissecting microscope. From each ovary slice, follicles of the correct size (1 mm) were dissected out of the slice, using 27-gauge needles to tease and tear out the follicles, and were washed in sterile PBS. Fifteen follicles from each ovary were pooled for each assay (in a volume of approximately 100 µl of PBS) and placed at-70°C until assayed, or fixed for flow cytometric analyses.

Granulosa cells from medium-sized antral follicles (3 mm) and large antral follicles (6–8 mm) Granulosa cells were gently scraped from medium-sized antral (3 mm) and large antral follicles (6–8 mm) of approximately 10–15 ovaries into separate tubes containing approximately 20 ml of sterile PBS. The cell suspension was centrifuged at 1000 rpm for 10 min, and the supernatant discarded. The cell pellet was placed at-70°C until assayed, or fixed for flow cytometric analyses.

Flow Cytometric Analyses

Follicle pools and granulosa cell pellets were resuspended in 750 µl of PBS, fixed by the drop-wise addition of 2 ml 70% ethanol (-20°C) while gently vortexing, and stored overnight at 4°C. Fixed cells were pelleted, washed twice in PBS with EDTA, and resuspended in propidium iodide (PI)-staining buffer (Hanks' balanced-salt solution containing 0.1% Triton X-100, 50 µg/ml PI, and 100 µg/ml DNase-free RNase) and protected from light until analyzed. DNA analysis was performed as described previously [24] using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) emitting a 488-nm beam. The FACScan was calibrated for linearity. Cellular debris and aggregated nuclei and clumps were omitted from the analyses by gating based upon the pulse area versus pulse width. Data from 10 000 nuclei were recorded, and the data were displayed as a frequency distribution histogram of the intensity of fluorescence emitted from PI at 575 nm.

In order to stain the nuclei for both Ki-67 and PI, the procedure described above for labeling with PI was used; the cell nuclei were then resuspended in PI-buffer containing a 1:500 dilution of Ki-67 antibody (monoclonal antibody to Ki-67 antigen clone MIB-1, from BioGenex, San Ramon, CA) and incubated for 1 h at room temperature. The nuclei were washed twice in PBS, then resuspended in a solution of 5-([4,6-dichlorotriazin–2-yl]amino)-fluorescein (DTAF)-conjugated goat anti-mouse IgG (1:500 dilution; Pharmingen, San Diego, CA), and incubated for 1 h at room temperature. After two final washes in PBS, the nuclei were resuspended in PI-buffer. The nuclei were then subjected to a flow cytometric analysis. The FACscan data were saved and displayed as a dot plot of the fluorescence intensity omitted from PI versus the fluorescence intensity of the DTAF fluorochrome. At least three samples were analyzed separately for each group of follicles.

Telomerase Activity

Telomerase activity was measured in follicle samples using a commercially available telomerase polymerase chain reaction (PCR) ELISA kit (Boehringer-Mannheim, Indianapolis, IN) by following the protocol supplied with the kit. The sample groups in which telomerase activity was measured were as follows: preantral follicles (60–100 µm), small antral follicles (1 mm), granulosa cells scraped from 3-mm medium-sized antral follicles, and granulosa cells scraped from 6–8-mm large antral follicles. Each sample group was prepared three independent times to obtain mean ± SEM values. The 293 cell line was used as a positive control for telomerase activity. To visualize the telomerase PCR products obtained from the ELISA kit, DNA samples were end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. The labeled samples were separated by electrophoresis on a 10% polyacrylamide gel and were visualized by autoradiography.

In Situ Hybridization

The plasmid pGrn83, containing 559 base pairs (bp) corresponding to the exact 5' and approximate 3' end of the RNA component of human telomerase (hTR) from the genomic clone (Geron Corporation, Menlo Park, CA), was used as a template to generate sense (hTR linearized with PstI restriction endonuclease, SP6 RNA polymerase) and antisense (hTR linearized with MluI restriction endonuclease, T7 RNA polymerase) RNA probes. Digoxigenin-labeled single-stranded RNA probes were synthesized using an RNA-labeling kit (Boehringer-Mannheim). The degree of incorporation of digoxigenin into the RNA probe was assessed by the level of alkaline phosphatase activity per amount of RNA synthesized. Transcripts were alkaline hydrolyzed for 21 min in 80 mM NaHCO3, 120 mM Na2CO3, and 20 mM ß-mercaptoethanol at 60°C to generate probes with an average length of 300 nucleotides for efficient hybridization.

Paraffin-embedded bovine ovarian tissue sections (5 µm) were cut and placed onto RNase-free, 3-aminopropyl-triethoxysilane (Sigma)-treated slides (with each section processed in parallel for antisense and sense RNA probes). The sections were heated (65°C for 60 min), deparaffinized in xylene, rehydrated with decreasing concentrations of ethanol (100% two times, 95%, 90%, 70%, and 50% each for 2 min), and treated with 20 µg/ml proteinase K (Boehringer-Mannheim) in a buffer of 50 mM Tris-HCl (pH 7.5) and 5 mM EDTA for 20 min at 37°C. After rinsing in PBS, the sections were postfixed in 4% paraformaldehyde/PBS, rinsed in PBS, dehydrated with increasing concentrations of ethanol (50%, 70%, and 100% each for 1 min), and desiccated before hybridization.

Sections were hybridized overnight at 50°C in a solution of 50% formamide, 10% dextran sulfate, 700 µg/ml Escherichia coli tRNA, 0.5% Triton X-100, 0.3 M NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 10 mM Na2HPO4 (pH 6.8), and single-strength Denhardt's solution containing digoxigenin-labeled RNA probes. The sections were washed three times, 45 min each time, at 50°C in a solution of 50% formamide, 0.5% Triton X-100, 0.3 M NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 10 mM Na2HPO4 (pH 6.8), and single-strength Denhardt's solution. The sections were rinsed (three times, 2 min each) in RNase buffer containing 0.4 M NaCl, 10 mm Tris-HCl (pH 7.5), and 5 mM EDTA (pH 8.0) and treated with 150 µg/ml RNase A at 37°C for 1 h.

After two 15-min rinses in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate)/0.5% Triton X-100 at 50°C, hybridized probe was detected using an alkaline phosphatase-conjugated anti-digoxigenin antibody from sheep (Boehringer-Mannheim). The sections were preblocked for 1 h in 5% milk powder in PBS, incubated with the anti-digoxigenin antibody (1:750) for 1 h at room temperature in a humidified atmosphere, rinsed with PBS (three times, 5 min each), and reacted with 450 ng/ml 4-nitro blue tetrazolium chloride (Boehringer-Mannheim) and 350 ng/ml 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer-Mannheim). The slides were counterstained with 0.075% methyl green and mounted with 80% glycerol in PBS under a glass coverslip; they were examined and photographed using a BX50 microscope (Olympus Optical, Tokyo, Japan).

Statistical Analyses

The relationship between follicle size and telomerase activity was analyzed by multiple regression analysis, using the Statistical Package for the Social Sciences (SPSS International BV, The Netherlands).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Cycle Analyses

Cell cycle analyses were performed to determine the proportions of cells within follicles at different stages of growth. The results obtained from cell cycle analyses of 60–100-µm preantral follicles, 1-mm small antral follicles, and granulosa cells from 3-mm medium-sized antral follicles and 6–8-mm large antral follicles are shown in Figure 1. Data in Figure 1 demonstrate that the highest proportion of cells (> 80%) within a follicle were in Go/G1, with only approximately 15% in S phase and a very small proportion actively dividing in G2/M phase. The proportions in G2/M in particular changed between follicle sizes. In addition, a distinctive peak for cells that were apoptotic was observed in larger antral follicles (Fig. 1D).



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  		FIG. 1. Representative cell cycle analyses from each of the pools of preantral follicles (A), small antral follicles (B), and granulosa cells from medium-sized antral follicles (C) and large antral follicles (D). The data are a frequency distribution (numbers) of the fluorescence intensity (channels) of nuclei recorded at the emission wavelength of PI (575 nm). The values are mean percentages ± SEM of nuclei in G0/G1, S, or G2/M, derived from three independent samples for each size of follicle.

We examined the expression of Ki-67, a protein expressed in dividing but not G0 cells [25]. We used dual staining and two-color flow cytometry to quantitate the expression of Ki-67 simultaneously with cell cycle analysis in 60–100-µm preantral follicles, 1-mm small antral follicles, and granulosa cells from 3-mm medium-sized antral follicles and 6–8-mm large antral follicles. The results are shown in Figure 2. The y-axes represent fluorescence intensity of the Ki-67 antibody staining, and the x-axes represent the stage of the cell cycle (PI staining of DNA). The results for cells stained with Ki-67 antibody, shown in red, versus cells stained with no primary antibody, shown in blue, demonstrated that only a small proportion of cells in the G0/G1 phase were expressing Ki-67 and hence dividing, and that most of the granulosa cells were therefore resting in G0 in all the follicle sizes examined.



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  		FIG. 2. Flow cytometric analyses of cell nuclei dual stained with Ki-67 and PI from preantral follicles (A), small antral follicles (B), and granulosa cells from medium-sized antral follicles (C) and large antral follicles (D). Each figure shows two dot plots (red and blue) of nuclei stained with a primary antibody to Ki-67 (red), or without a primary antibody (blue), using DTAF-conjugated secondary antibody. The y-axes represent log of fluorescence intensity of the DTAF fluorochrome, and the x-axes the intensity of PI fluorescence.

Telomerase RNA Component

Detection with in situ hybridization The telomerase RNA component was localized in the ovary using in situ hybridization (Figs. 3–5). Positive staining was readily detected in the granulosa cells of growing follicles. The surface epithelium and stromal tissue were negative (Fig. 3A). In bovine ovaries, primordial follicles are located 0.1–0.5 mm from the surface of the ovary (Fig. 3A). Each primordial follicle consists of an oocyte surrounded by nondividing inactive granulosa cells [26]. No staining for the telomerase RNA component was apparent in the primordial follicles encountered (Fig. 3, A and D). Primary follicles, which have a single layer of cuboidal granulosa cells surrounding the oocyte, exhibited staining for the telomerase RNA component only in the granulosa cells themselves. No staining was visible in the oocyte at this stage of follicle development. Preantral follicles also stained strongly for telomerase RNA component in the granulosa cell population only (Fig. 3, A–D). Antral follicles displayed strong staining of granulosa cells in all regions of the follicle and also of cumulus cells surrounding the oocyte as shown in Figure 4, A and B. The controls, in which sense RNA was used for hybridization, did not have any specific staining (Fig. 4C). In large healthy (Fig. 5, A–D) and atretic (Fig. 5E) antral follicles, positive staining for the telomerase RNA component was observed in the membrana granulosa. The distribution of staining of telomerase RNA component varied throughout the granulosa cell layers from follicle to follicle, and generally not all cells stained positively. The most common pattern observed was positive staining in the middle (Fig. 5A) and antral layers (Fig. 5B). Less frequently, staining in the basal layers of granulosa cells was observed (Fig. 5C). Positively stained cells that were randomly dispersed throughout the membrana granulosa, as shown in Figure 5D, were also observed in some follicles. The control sections hybridized with sense RNA did not have any specific staining (example shown in Fig. 5F).



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  		FIG. 3. Light micrographs of in situ hybridization of the digoxigenin-labeled telomerase RNA component (hTR probe) to bovine ovaries, detected as a positive alkaline phosphatase reaction (shown as a black precipitate). No staining of primordial follicles (arrowheads) situated close to the surface (s) of the ovary was observed (A). Granulosa cells (g) of preantral follicles (A–D) (arrows) stained positively. No staining was evident in oocytes (o). Bars = 30 µm (A, D) or 20 µm (B, C). Sections in C were photographed with Nomarski differential interference optics.



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  		FIG. 4. Light micrographs of in situ hybridization of the digoxigenin-labeled telomerase RNA component (hTR probe) to bovine ovaries, detected as a positive alkaline phosphatase reaction (shown as a black precipitate). In antral follicles, granulosa cells and cumulus cells (arrows) stained positively, but oocytes (o) were negative (A, B). C) A section equivalent to that shown in B but hybridized with sense RNA probe as a negative control. Bars = 20 µm (A) or 30 µm (B, C). Antrum (a). Sections in B and C were photographed with Nomarski differential interference optics.



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  		FIG. 5. Light micrographs of in situ hybridization of the digoxigenin-labeled telomerase RNA component (hTR probe) to bovine ovaries, detected as a positive alkaline phosphatase reaction (shown as a black precipitate). In healthy (A–D) or atretic (E) antral follicles, a variable pattern of positive staining (arrow) was observed in the membrana granulosa. In each figure the antrum is uppermost; "g" indicates the middle of each membrana granulosa. A healthy antral follicle hybridized with a sense RNA probe as a negative control is shown in F. Bars = 20 µm (A–C, F) or 30 µm (D, E). Sections in C, D, and F were photographed with Nomarski differential interference optics.

Telomerase Activity

We utilized a PCR-based telomerase ELISA assay to determine the level of telomerase activity in cells from follicles of various sizes including 60–100-µm preantral follicles, 1-mm small antral follicles, and granulosa cells from 3-mm medium-sized antral follicles and 6–8-mm large antral follicles (n = 3 pools of follicles or cells per group). Telomerase activity was inversely related to follicle size (p < 0.05, linear regression), being highest in the smallest follicles (60–100 µm) and declining as the follicles enlarged to 3 mm, where the level remained low through to a follicle size of 6–8 mm (Fig. 6B). In this telomerase ELISA assay, the telomerase activity was not related to the amount of enzyme but to the log of the amount of enzyme, as demonstrated by assaying the activity of increasing numbers of 293 cells (Fig. 6A). Therefore, the results in Figure 6B substantially underestimate the amount of telomerase activity in the smaller follicles. Furthermore, the presence of telomerase activity was confirmed by end-labeling the PCR products from the ELISA assay to demonstrate the distinctive telomerase-specific repeat ladders (data not shown).



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  		FIG. 6. Telomerase activity in 293 cells in increasing concentrations (A) and in preantral (60–100 µm) follicles, small antral (1 mm) follicles, and granulosa cells from medium-sized antral (3 mm) and large antral (6–8 mm) follicles (n = 3; mean ± SEM) (B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For the first time, we have shown that the granulosa cells and not the oocyte of the ovarian follicle are the source of the telomerase activity in the ovary. Our results were based on mRNA in situ hybridization, and localizing the telomerase RNA component to granulosa cells and not oocytes. We have also demonstrated telomerase activity in follicles, the highest levels being found in the smaller preantral follicles. These results support our previous hypothesis [14–18] that granulosa cells in mature ovarian follicles are derived by division and differentiation of a population of stem cells.

It has been previously estimated that the numbers of granulosa cells double 21 times, increasing from 24 to 40 x 106 during bovine follicular growth and development [26]. Existing and current evidence suggests that during the course of follicular development, growth is simply not due to 21 successive rounds of cell division of all granulosa cells. For this to be so, it could be expected that the proportion of cells dividing and the mitotic index would remain constant during follicular growth, and mitotic figures would be evenly distributed throughout the membrana granulosa. Evidence suggests that this is not the case. In the current study we found that the proportion of cells in G2/M varied with follicular size. It has already been observed that the mitotic index changes during the course of follicle growth [27–29]. Mitotic activity of granulosa cells has also been found to be higher in cells close to where the oocyte is attached to the follicle wall [30] and in the other regions of the bovine antral follicle wall; more than three quarters of mitosis occurs in the middle layers of the multilayered membrana granulosa (unpublished results). In addition, the length of doubling time has been reported to change during follicular development [29]. All of these observations of the in vivo situation clearly show that not all granulosa cells are of equal potential for division.

It is from these and other observations that we postulated that during follicular growth granulosa cells arise from a population of stem cells [14–18], namely committed stem cells. Functional properties of stem cells in general include pluripotency, lack of inhibition of mitosis due to cellular contact, the ability to divide without anchorage in vitro, and, from recent studies, expression of telomerase [8–11]. We have previously suggested [13] that lack of contact inhibition in vivo occurs during growth of a follicle, particularly at the preantral stages in which granulosa cells are in close physical contact, and in antral follicles where cells divide within the layers of the membrana granulosa [31]. In addition, when cultured on a solid substratum, granulosa cells can readily form aggregates or hills [32], and a small fraction (< 5%) of granulosa cells can divide and form colonies when cultured without anchorage [14–18]. The pluripotential nature of granulosa cells has not fully been addressed. Currently, three subtypes are recognized as derived from precursor epithelioid cells of the primordial follicle—cumulus cells that surround the oocyte, and in the other area of the follicle the cells in the basal and antral layers. The cells in the basal and antral layers apparently differ in the level of expression of many genes [33–37] and consequently are regarded as subtypes of the granulosa cell. Studies using chimeric transgenic animals suggest that basal and antral cells can be derived from the same progenitor cells [38] and therefore were derived from pluripotent cells. In the current study we now have additional evidence that there exists a population of stem cells in the membrana granulosa. We detected both telomerase activity and the RNA component of telomerase in granulosa cells in follicles of various sizes.

Additional support for the stem cell characteristics of granulosa cells comes from cloning by nuclear transfer from somatic cells. Transfer using donor nuclei from a mammalian cell line [39, 40] or cumulus cells [41] had overall success rates of less than 0.4% and 0.9%, respectively; no clones were obtained from neuronal or Sertoli cells [41]. However, using mural granulosa cells from cattle, overall success rates of 2.8% have been achieved (D.N. Wells, personal communication). Although cloning technologies are in their infancy, it is clear from research in nonmammals that embryonic development via nuclear transfer is more successful the less differentiated the cells providing the donor nuclei are [42]. Thus, if the same is true in mammals, the success of cloning from granulosa cells is due, partly at least, to the stem cell character of some of the granulosa cells.

From our hypothesis it is predicted that the greatest concentration of stem cells would be found in the primordial follicles. However, we did not detect the telomerase RNA component in the pregranulosa cells of these follicles. These cells have remained quiescent since fetal life and thus have had no immediate need for telomerase. In the hematopoietic system, the resting stem cells also do not express telomerase [43]. Therefore it appears that a key component of activation of stem cells includes activation of telomerase. Furthermore, it is also predicted that the proportion of granulosa cells that are stem cells would progressively decline as non-stem cell progeny accumulate. Assuming constant telomerase activity in the stem cells, it would be expected that the telomerase activity, averaged out over the total number of granulosa cells in a follicle, would thus decline as the follicle increased in size. In our studies in the cow, and that of Eisenhauer et al. [23] in the rat, this was observed to be the case, although in the latter study the activity was attributed to the oocyte.

One major difference between the membrana granulosa and other epithelia such as the skin is that granulosa expands laterally, and that the layers of cells increase from one layer to about 6–8 layers. Lateral expansion occurs as the primordial follicle is activated and begins to grow, from about 30 µm to about 2 cm in diameter, representing about 19 doublings in surface area (calculated from [26]). Thus there is the potential for both lateral movement, as occurs in gut epithelium, and movement in the basal/antral direction, as occurs in skin epithelium. Unlike the situation in skin, in which stem cells are reported to exist in the most basal layer, the most mitotically active cells in the membrana granulosa of antral follicles were observed in the middle layers (unpublished results). Therefore the more mature cells exist toward the basal lamina and toward the antrum. It would have been expected then that telomerase would be highest in the middle region if some of the replicating cells were stem cells, and lowest in the most basal and most antral regions of the membrana granulosa. However, we observed that many layers had detectable telomerase RNA component. Most follicles had telomerase RNA in the middle and antral layers and others had telomerase RNA in the middle and basal layers. We suspect that not all these cells are stem cells, but rather stem cells and their most immediate progeny. From the only study of the regulation of both the telomerase catalytic and RNA components [44], it would appear that only the telomerase catalytic, and not the RNA component, is acutely negatively regulated during differentiation. Thus the differentiating progeny of stem cells could have no telomerase activity but still have residual telomerase RNA.

The different telomerase RNA-staining patterns of antral follicles observed indicate that the follicles are of at least two types. In one type the younger progeny progress from the middle dividing layers toward the antrum. In the other they progress from the middle layers in the opposite direction toward the basal lamina. From our other studies (unpublished results), three other features of follicles that classify bovine antral follicles into two types have been observed: first the ultrastructure of the basal lamina; secondly the shape of the basal granulosa cells in relation to the overall structure of the membrana granulosa; and thirdly the process of atresia, particularly the location (basal or antral) where cell death and degeneration take place first. The significance of these observations is not readily apparent at this stage.

There are now conflicting results on whether telomerase is present in mammalian oocytes, and our study may help clarify the discrepancy. In the male gonad, telomerase is expressed in the germ cells of the human [21] and the rat [23]. In the female gonad it has been reported that telomerase is expressed in the germ cells of Xenopus [22] and the rat [23]. In contrast to the finding in the rat, there are two other mammalian species, the human [6] and cattle (current results), in which telomerase has not been detected in oocytes. Germ cells in male gonads, and also in female gonads of nonmammalian species such as Xenopus, replicate, so one could predict that telomerase is expressed in the germ cells of these gonads. Oocytes in mammalian follicles do not replicate; therefore the need for telomerase is not apparent. Eisenhauer et al. [23] concluded that rat oocytes express telomerase; the rat oocytes were isolated by pipetting from dispersed follicles, and telomerase was then measured by a sensitive PCR-based telomeric repeat amplification protocol [21]. It is possible that the telomerase-rich granulosa cells may have contributed to the positive signal that the investigators observed and interpreted as due to oocytes. Clearly further studies are needed to determine whether the rat oocyte does or does not express telomerase.

In summary, the expression of the telomerase RNA component in bovine ovaries is not associated with germ cells as it is in the testis. This concurs well with the general lack of division of oocytes in mammalian ovaries until just prior to ovulation when meiosis resumes. Our results also support the hypothesis that primordial follicles have resting stem cells that on activation of follicles express telomerase, divide, and contribute to the mature populations of cells seen in antral follicles. Whether only stem cells divide, or whether an additional transit-amplifying population of more differentiated but dividing cells exists, as occurs in the crypts of the luminal gut epithelium and hair follicles, remains to be determined.


   ACKNOWLEDGMENTS
 
The authors would like to thank Ingrid van Wezel and Masashige Kuwayama for their help and technical advice, and the Geron Corporation for their generous gift of the plasmid pGrn 83.


   FOOTNOTES
 
1 This work was supported by the National Health and Medical Research Council of Australia, Flinders University of SA, and Flinders Medical Centre Research Foundation. Back

2 Correspondence: Raymond J. Rodgers, Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia. FAX: 618 8204 5450; ray.rodgers{at}flinders.edu.au Back

Accepted: March 5, 1999.

Received: July 30, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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