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Ovary |
Prince Henry's Institute of Medical Research,3
Department of Biochemistry and Molecular Biology,4 Monash University, Clayton, Victoria 3168, Australia
MRC Human Reproductive Sciences Unit,5 Centre for Reproductive Biology, The Chancellors Building, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom
Centre for Urological Research,6 Monash Institute of Reproduction and Development, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, follicle, follicular development, mechanisms of hormone action, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the candidates regulating the size and activation of the pool of primordial follicles are increased levels of LH and Mullerian inhibiting substance (MIS), which have been associated with decreased numbers of germ cells, primordial follicles, or both [8–11]. On the other hand, MIS-null mice exhibit enhanced loss of primordial follicles that is consistent with a role of MIS in regulating activation of primordial follicles [10, 12]. Changes in the size and proliferation of pregranulosa cells are hallmarks of the transition of primordial follicles to actively growing follicles, suggesting that factors influencing cell cycle regulation may be important. Proliferating cell nuclear antigen (PCNA) can be used as an early marker of granulosa cell proliferation [13, 14]. The oocyte-specific factor, growth differentiation factor 9 (GDF9), has been shown to mediate communication between the oocyte and surrounding granulosa cells in early follicles and to increase primordial follicle activation and growth [15, 16], while the zinc finger containing DNA-binding protein, Wilms tumor 1 (WT-1), is essential for ovarian formation in mice [17, 18] and may have a role in regulating the primordial follicle pool.
A role for estrogen (E2) in primordial follicle formation and activation has not been demonstrated in the rodent. Models of estrogen receptor (ER) deficiency have not shown a block in follicle development until the antral stage (for a review see [19]), however, quantitative measures of primordial follicle numbers have not been reported in these models. A role for E2 is plausible considering the presence of ER within developing ovaries, which possess only primordial and primary follicles [20]. The simultaneous activation of postnatal follicles and the drop in elevated E2 (5 x 10–7M at 4 h after birth, to 2 x 10–8 48 h after birth) suggest that E2 may be important in the early stages of follicle formation and development in rodents [2]. It is also possible that E2 may have indirect effects on primordial follicles through factors such as those described above, which are known to influence follicle formation and activation.
Female aromatase knockout (ArKO) mice are deficient in E2 and offer an excellent animal model for testing the effects of E2 on follicle formation and early development. They have a block in follicular development at the antral stage and possess Sertoli cell-like cords. We have previously shown that E2 is not required for the initiation of follicle growth, but is necessary for the later stages of follicular development in ArKO mice [21]. However, we have not quantified the numbers of primordial follicles in ArKO mice versus wild-type mice. Assessing the expression profile of the genes for factors such as those mentioned above and postulating the influence of early follicle formation and activation in female ArKO mice may reveal direct or indirect actions of E2 on primordial follicle endowment.
The overall objective of these studies was to define the role for E2 in primordial follicle formation and activation by assessing the numbers of primordial and primary follicles. We also tested MIS and WT-1 protein and GDF9 mRNA expression in ArKO versus wild-type ovaries, because these factors have been implicated in follicle activation. PCNA staining in the ovaries of wild-type and ArKO mice was also compared as an index of cell proliferation. To further assess the role of E2, we quantified the numbers of primordial and primary follicles following E2 treatment of ArKO mice and examined the levels of MIS, WT-1, and PCNA protein and GDF9 mRNA expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wild-type and ArKO mice on a J129/C57B6 background [21] were maintained under specific pathogen-free (SPF) conditions, on a 12L:12D regimen and fed ad libitum a soy free mouse chow (Glen Forrest Stockfeeders, Western Australia), with undetectable levels of isoflavones. All animal procedures were approved by the Animal Ethics Committee at Monash University and were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Tissue Collection and Processing
Daily vaginal smears were taken and stained using Diff Quick Stain (Lab Aids, Narrabeen, Australia). ArKO mice are acyclic [21] with vaginal smears showing persistent diestrus. All ovaries of wild-type and ArKO mice treated with E2 were collected at the end of the 21-day treatment, because these mice have vaginal smears indicative of persistent estrus. The ovaries from placebo-treated ArKO mice were collected at the end of the treatment period, because these mice do not cycle. Placebo-treated wild-type mice were collected in diestrus. Animals were killed at 6, 10, or 16 wk of age by cervical dislocation and one ovary from each animal (n = 5–6/group) was immersion-fixed in Bouins fluid. Fixed tissue was processed through a graded series of alcohols and embedded in paraffin wax. Serial 3-µm sections were cut, and every 10th and 11th section pair was stained with a modified Mason trichrome stain [22]. Intervening sections were kept for immunohistochemistry.
Estrogen Treatment
Subcutaneous pellets containing either 0.05 mg of 17ß-estradiol or placebo (Innovative Research of America, Sarasota, FL) were administered for 21 days to 7-wk-old wild-type and ArKO mice (maintained on a soy-free diet) as previously described [23]. Treatment was performed at this age because the development of Sertoli-filled cords begins in the ovaries of ArKO mice at this time [24]. The E2 pellets restored serum estradiol to 50–100 pg/ml (peak estrous levels) and increased uterine weights of ArKO mice to wild-type levels.
Morphological Classification of Small and Growing Follicles
Follicles were classified as described previously [25]. In brief, follicles were classified as primordial if they contained an oocyte surrounded by a complete layer of squamous granulosa cells. Follicles that included some cuboidal granulosa cells but still had a majority of squamous granulosa cells were also classified as primordial (also called transitory primordial follicles). Primary follicles showed a single layer of cuboidal granulosa cells; follicles that had both cuboidal and squamous granulosa cells, in which cuboidal cells predominated, were classified as primary. Many previous investigations used the Pedersen and Peters [26] follicle classification scheme, which determines class based on granulosa cell number rather than follicle and granulosa cell appearance. In the present study, we have classified follicles according to the type (squamous or cuboidal) and number of granulosa cell layers, which correspond to types 1 to 3b described by Pedersen and Peters [26]. Secondary follicles possessed two or more layers of cuboidal granulosa cells without an antrum. Antral follicles contained fluid-filled spaces within their granulosa cell layers. They were classified as small antral if they possessed less than five granulosa cell layers, and as large antral follicles when the granulosa cell layers exceeded five layers. Multioocyte follicles contained more than one oocyte enclosed within the granulosa cell layer or layers.
Quantification of Early Follicles
A fractionator/physical dissector design [27, 28] was used to estimate follicle number in the ovaries of wild-type and ArKO ovaries obtained at 10 wk of age, as has been described previously for wild-type mice [25]. Briefly, follicle counts were made using the CASTGRID system (v2.1.4, Olympus Danmark A/S, Albertslund, Denmark) and a stereological design incorporating the fractionator and physical dissector. Estimates of follicle number per ovary were made by incorporating stereological sampling protocols on a predetermined fraction of ovarian serial sections. In this case, every 10th and 11th sections were used, and follicles (with visible nuclei) were counted if they appeared in one section but not the consecutive "look up" section, which ensures follicles are counted only once.
Oocyte and Oocyte Nuclear Diameters
Oocyte and oocyte nuclear diameters associated with all counted follicles were measured (n = 40–80 follicles/ovary). Profiles of oocytes and oocyte nuclei were measured by calculating the geometric mean of the long and short axes measured using the CASTGRID system.
Immunohistochemistry
For immunohistochemical analysis, four sections per ovary for each of three animals per genotype and treatment were used. Negative controls used in each study consisted of either normal serum from the species in which the primary antibody was raised (WT-1 and MIS) or immunoglobulin G (IgG)-matched negative preimmune serum (PCNA). Staining was assessed using a semiquantitative intensity classification. Staining was either absent (–), slight (+), or moderate (++). The positive controls for the MIS and WT-1 antibodies were immature Day 1 testis and adult wild-type ovary, respectively [29–32].
Mullerian Inhibiting Substance
Paraffin-embedded sections were subjected to immunohistochemical staining for the MIS protein (also referred to as AMH) as described in detail in [33]. Following dewaxing and rehydration, slides were incubated in 3% (v/v) hydrogen peroxide in methanol. Slides were washed in Tris-buffered saline (TBS) 0.05 mol/L (pH 7.4) and 0.85% NaCl and blocked in TBS containing 5% BSA (Sigma) prior to addition of the primary antibody. Primary MIS antibody (goat anti-MIS; Santa Cruz Biotechnology, Santa Cruz, CA) raised again human MIS (the sequence differs from mouse by a single amino acid) was used at 1:500, and slides were incubated at 4°C overnight. Slides were incubated with rabbit anti-goat secondary antibody conjugated to biotin at a dilution of 1:500 (rabbit anti-goat, Vector Labs, Burlingame CA). The biotinylated antibody was linked to horseradish peroxidase (HRP) by a 30-min incubation with an avidin-biotin-HRP complex (ABC-HRP; DAKO A/S, Glostrop, Denmark). Protein localization was then determined using diaminobenzidine (liquid DAB; DAKO). Slides were counterstained in hematoxylin, dehydrated, and mounted using Pertex mounting media (Cell Path Hemel, Hempstead, UK).
Wilms Tumor-1
Following dewaxing and rehydration, slides were subjected to citrate buffer antigen retrieval (microwave 5 min), and blocked in 3% (v/v) hydrogen peroxide in methanol. Tissue was further blocked in normal goat serum, TBS (pH 7.4) containing 5% BSA (Sigma), and avidin (4 drops/ ml). Finally, a biotin in TBS blocking step was also used.
Primary antibody goat anti WT-1 (DAKO) (amino acids 1–181 of human WT-1), was used at 1:500 and slides were incubated at 4°C overnight. Slides were incubated with rabbit anti-goat secondary antibody conjugated to biotin at a dilution of 1:500 (Vector Labs, Burlingame CA). The biotinylated antibody was linked to HRP by a 30-min incubation with ABC-HRP (DAKO). Protein localization was then determined using liquid DAB (DAKO). Slides were counterstained in hematoxylin, dehydrated, and mounted using Pertex mounting media (Cell Path Hemel, Hempstead, UK).
Proliferating Cell Nuclear Antigen
Proliferating cells were identified using monoclonal antibody to PCNA (PC10; DAKO). Before immunolocalization was performed, sections were rehydrated, then exposed to antigen retrieval (0.01 M citrate buffer pH 6.0, boiled 10 min). Subsequent immunostaining was performed using an automated DAKO Autostainer (DAKO, Carpinteria, CA). Briefly, sections were incubated with 0.03% H2O2 (DAKO) for 15 min to quench endogenous peroxidase. Sections were then treated with CAS-block (Pierce, Rockford, IL) for 30 min to block nonspecific binding before being incubated with primary antibody (1:1200, 30 min) or concentration-matched negative control mouse IgG2a. Primary antibodies were detected by 15-min incubation with biotinylated rabbit anti-mouse IgG2a and an avidin-biotin peroxidase kit (ABC-Elite, Vector Laboratories) for 15 min. Immunostaining was visualized by reaction with DAB (DAKO) for 5 min. Sections were counterstained with 0.1% Mayers hematoxylin, dehydrated gradually with alcohol, cleared with histolene, and mounted under DePeX (BDH Laboratory Supplies, Poole, England) before quantification of protein marker expression.
Gene Expression
The expression levels of GDF9 mRNA in ovaries of wild-type and ArKO mice treated with placebo or E2 were measured. RNA was extracted from individual ovaries using a phenol-chloroform based method (Ultraspec, Fisher Biotech) incorporating an additional chloroform extraction step, and followed by DNase treatment. RNA was reverse transcribed using random primers (Roche, Mannheim, Germany) and AMV reverse transcriptase (Promega, Madison, WI). Complementary DNA was diluted 1: 20 and amplified by real-time polymerase chain reaction (PCR) (Roche) using Fast Start Master SYBR Green I (Roche), and specific oligonucleotide pairs. All PCR products exhibited a single peak in melting curves and were identified as single bands of the appropriate size on ethidium bromide gels. Amplified products were verified with sequencing. Experimental samples were quantified by comparison with cDNA standards of known concentration (0.01–1000 fg/µl). We have expressed all RNA transcript levels relative to 18S because it was the most reproducible housekeeping gene and had acceptable variation within genotype (coefficient of variation less than 10%). Three separate reverse transcription (RT) reactions per sample were subjected to PCR analysis and then the mean value was calculated from the three RTs, each corrected for 18S. For each separate RT on a group of age-matched ovaries, all mice across genotype and treatment were analyzed in a single PCR. This allows comparisons of expression to be performed across genotype and treatment.
Data are represented as relative levels of expression per amount of RNA. Transcript levels for GDF9 were assessed using in-house designed primers against mouse GDF9: forward, 5'-GAC CGC TCC ATC GCT TAC AAA-3'; reverse, 5'-CAC ACT TCC CCC GCT CAC A-3' (193 base pairs).
Statistical Analysis
Data are presented as the mean ± SEM. Statistical analysis of follicle number was performed using Sigmastat statistical software (version 2.0; Jandel Corporation, San Rafael, CA). If data were normally distributed, they were subjected to two-way analysis of variance (ANOVA). If data were not normally distributed they were log transformed and then subjected to two-way ANOVA. If genotype or treatment had a significant effect on follicle number, diameter, or gene expression, multiple pairwise comparisons were made using a Tukey test. If no effect was observed between treatments within the same genotype, for graphical purposes, data were pooled and genotypes were compared. A value of P < 0.05 was considered significant.
Statistical analysis of oocyte and oocyte nuclear diameters were performed using Minitab statistical software (Minitab Inc., State College, PA). An ANOVA was performed on data using the mouse as a random factor nested within treatment. As the number of observations per animal were not equal across the groups studied, the diameters of the oocytes measured in each mouse were averaged, ensuring unbiased variance. Although averaging the values for each mouse leads to some lack of homogeneity in the standard deviations, because the number of observations per mouse are not the same, the Levene and Bartlett test for heterogeneity of variance is not significant (P = 0.703, P = 0.442). A two-way ANOVA was then performed, followed by a Tukey posthoc test defining the differences in genotype and treatment. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicle number per ovary Numbers of primordial follicles (Table 1) were decreased in ArKO mice compared with wild-type mice (P = 0.034), and primary follicle numbers (Table 1) showed a decrease that was approaching significance (P = 0.062). No significant effect of E2 treatment was observed.
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Oocyte diameters—primordial follicles Despite a high variability between mice among each group (same genotype and treatment) diameters of oocytes in primordial follicles (Table 2) were increased in placebo-treated ArKO mice compared with placebo-treated wild-type mice (P < 0.05), and oocyte diameter was decreased in both wild-type and ArKO mice treated with E2 (P = 0.015) compared with placebo treated, genotype matched controls (P < 0.05). The diameters of primordial oocyte nuclei (Table 2) were increased in placebo treated ArKO placebo versus wild-type mice, although the variance between mice masked the significance of these differences (P = 0.091). Again, E2 treatment in both wild-type and ArKO mice decreased oocyte nuclear diameter compared with their placebo counterparts, although the variance between mice masked the significance of these differences (P < 0.057). Using the diameters of the oocytes described above, the oocyte volumes were calculated. The oocyte volume in ArKO follicles was increased by 13% compared with placebo-treated wild-type controls, and was decreased by 9% in ArKO + E2 compared with ArKO + placebo. Wild-type + E2 oocyte volumes were decreased by 7% compared with those of wild-type + placebo. Similarly, the nuclear oocyte volume in ArKO oocytes was increased by 16% compared with placebo-treated wild-type controls. The oocyte nuclear volumes were decreased by 19% in ArKO + E2 mice compared with ArKO + placebo mice. In wild-type + E2 animals, oocyte nuclear volume was decreased by 5% compared with wild-type mice + placebo.
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Oocyte diameters—primary follicles No variability among mice within each group (same genotype and treatment) was noted for oocyte, or oocyte nuclear diameter measurements within primary follicles. When oocyte diameters (Table 2) were tested, differences were found among the four treatments (P < 0.001), the only significant pair being an increase in oocyte diameter in E2-treated ArKO mice compared with placebo-treated wild-type mice (Tukey posthoc test, P = 0.05). Oocyte nuclear diameters (Table 2) were similar in wild-type and ArKO mice, both treated and untreated. A comparison of the data in Table 2 shows that both oocyte and oocyte nuclear diameter increased as follicles moved from primordial to primary, irrespective of genotype or E2 treatment. Table 3 summarizes the mean percentage changes in oocyte and oocyte nuclear diameters induced by E2 treatment. The change was greater in ArKO mice than in wild-type mice for both primordial and primary follicles.
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The volume of oocytes in primary ArKO follicles was increased by 7% compared with wild-type, placebo-treated controls, and was increased by 22.5% in ArKO + E2 compared with ArKO + placebo. Volumes of wild-type + E2 follicles were also increased by 11% compared with those of the wild-type placebo. Similarly, the nuclear oocyte volume in ArKO follicles was increased by 8% compared with that of placebo-treated wild-type controls. The oocyte nuclear volumes were decreased by 3% in ArKO +E2 compared with ArKO + placebo. E2 treatment had no effect on oocyte nuclear volumes in wild-type follicles.
Multioocyte follicles The numbers of primordial multioocyte follicles (MOFs) as a percentage of total primordial follicles were similar in wild-type mice treated with either placebo (5.4%), or E2 (4.3%) and ArKO mice treated with either placebo (4.8%) or E2 (5.9%). The numbers of primary stage MOFs was similar in wild-type mice treated with either placebo (2.4%) or E2 (5.7%). There were no significant differences in the numbers of primary stage MOFs observed in ArKO mice treated with either placebo (8.1%) or E2 treatments (7.6%), or between ArKO and wild-type mice.
Mullerian Inhibiting Substance
No MIS staining was observed in primordial follicles of either genotype (data not shown). In 10-wk-old wild-type ovaries, MIS staining was observed in the granulosa cells of primary and secondary follicles (Fig. 1, A and B), whereas large antral follicles did not show MIS expression (Fig. 1C). The interstitial region of wild-type ovaries was largely negative for MIS staining (Fig. 1, A–C). The primary and secondary follicles of ArKO ovaries stained positively for MIS (Fig. 1, D and E), whereas antral follicles were negative (Fig. 1F). The Sertoli-filled cords, which predominated in 16-wk-old ArKO ovaries, did not stain positively for MIS (Fig. 1, G and H), although the Leydig cells surrounding these cords showed intense MIS staining (Fig. 1, G and H).
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Compared with wild-type ovaries (Fig. 2A), ArKO ovaries (Fig. 2B) showed increased intensity of MIS expression postpubertally. At 16 wk of age, the ovaries of ArKO mice had few if any antral follicles, and only a few preantral follicles; consequently, the extent of MIS expression appeared lower (Fig. 2C). Expression of MIS in 10-wk-old ArKO mice treated with estrogen was increased compared with placebo-treated ArKO mice (Fig. 2D). The intensity of immunostaining of MIS in 10-wk-old placebo treated wild-type and ArKO mice is summarized in Table 4.
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Wilms Tumor-1 (WT-1)
Wild-type ovaries (16 wk of age) showed WT-1 localization in preantral and small antral follicles (Fig. 3A). WT-1 localization was present in the granulosa cells of all follicles from the early primary and secondary stages of maturation (Fig. 3B) through the early antral stage (Fig. 3C). Large antral follicles of wild-type ovaries did not possess WT-1 protein (Fig. 3C), although the luteal cells of the corpus luteum did stain positively for WT-1 (Fig. 3D). The follicles of the ArKO ovary (16 wk of age) also stained positively for WT-1 (Fig. 3E) from the early primary stage of development (Fig. 3F). The adult ArKO ovaries did not possess many growing follicles, and many of the follicles that were present were transforming (Fig. 3G) into Sertoli-filled cords (i.e., they possessed both Sertoli and granulosa cells), or had undergone transformation and now appeared as Sertoli-filled cords (Fig. 3H); each stained positively for WT-1 protein. The intensity of staining for WT-1 protein was greater in ArKO ovaries (Fig. 3E) than in wild-type ovaries (Fig. 3A). The staining patterns of WT-1 in untreated wild-type and ArKO ovaries are summarized in Table 4.
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Proliferating Cell Nuclear Antigen
Overall, the intensity of PCNA staining was increased (more cells stained) in the ovaries of 16-wk-old wild-type mice (Fig. 4A) compared with age-matched ArKO mice (Fig. 4D), consistent with the atresia and transformation, which occurs in ArKO ovaries. Those early follicles that are present do not appear to show decreased PCNA staining in ArKO mice; rather, the presence of hemorrhagic cysts and Sertoli-filled cords in place of preantral and antral follicles accounts for the reduced PCNA staining (Fig. 4, B and C vs. E and F).
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The localization of PCNA-stained granulosa cells in primordial and primary follicles was similar in wild-type and ArKO mice. The primordial follicles of wild-type ovaries showed positive PCNA staining in squamous pregranulosa and cuboidal granulosa cells of primordial follicles (Fig. 5, A and B), and cuboidal granulosa cells of primary follicles (Fig. 5B). The primordial follicles of ArKO ovaries also showed PCNA staining in squamous and cuboidal granulosa cells (Fig. 5, C and D). The staining patterns of PCNA in untreated wild-type and ArKO mice are shown in Table 4.
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Growth Differentiation Factor 9
The relative expression level of GDF9 mRNA (Fig. 6) was significantly increased in 10-wk-old ArKO compared with age-matched wild-type animals (P < 0.001). No significant effect of E2 treatment was observed, although there was a trend for E2 treatment to decrease GDF9 mRNA levels in ArKO mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The decrease in primordial and primary follicles in the ovaries of ArKO compared with wild-type mice may be due to either 1) fewer primordial follicles forming from germ cell clusters, 2) increased activation of primordial follicles such that more primordial follicles enter the growing pool of follicles, or 3) earlier initial activation of primordial follicles in ArKO compared with wild-type mice, or a combination of the above.
Primordial Follicle Formation from Germ Cell Clusters
Consistent with the decreased number of primordial and primary follicles observed in ArKO ovaries, a decrease in the number of primordial follicles has been observed in embryonically E2-deficient baboons [34]. The E2-deficient baboons displayed a concurrent increase in the number of germ cell clusters, suggesting that the number of primordial follicles forming from germ cell clusters was decreased [34]. The proportion of MOFs in the primordial pool can be used as an index of the degree to which the process of follicle formation may be compromised. There were no differences in the incidence of MOFs between ArKO and wild-type mice in this study. It is possible, however, that an effect of E2 on MOFs in ArKO ovaries compared with wild-type mice was masked due to the background strain of mouse used, C57/BL6, which is known to have a high frequency of MOFs compared with other strains [35, 36]. Furthermore, the number of double oocyte primary follicles counted in the current study was only a small fraction (2.4%–8%) of the total number of primary follicles for which the counting protocol was optimized, suggesting that a more appropriate counting system may provide a more reliable estimate of double oocyte abundance. When 7-wk-old ArKO mice were treated with E2 in vivo, well after the time of follicle formation, no significant effects on the size of the primordial and primary follicle pools were observed, which is consistent with E2 playing a role in the formation of primordial follicles.
Follicle Activation
In cyclic females, a subset of follicles is activated each day and progresses to the primary stage with each cycle. An accelerated loss of primordial follicles from the resting pool has been observed during the initial wave of follicle growth in infantile rodents [37] and in perimenopausal women [38, 39], both situations associated with elevated gonadotropins compared with the reproductive years. Conversely, hypophysectomized rodents show a decrease in the initial recruitment as evidence by the larger resting pool compared with nonoperated controls [40, 41]. LH and FSH levels in ArKO mice are significantly elevated compared with wild-type counterparts [21, 42], which could mediate the reduction of primordial follicles in ArKO mice. LH has been associated with a reduction in the pool of primordial follicles; LH overexpressing mice display a 45% reduction in primordial follicles by 5 wk of age [11], which has been suggested to result from increased recruitment into the growing pool [11]. A role for estrogen in follicle activation was observed when comparing the rate of primordial follicle activation in culture, which is increased to 70% from the 30% observed in vivo, a phenomenon that has been postulated to occur due to the absence of the influence of circulating steroids [43]. However, in this study, E2 treatment of 7-wk-old ArKO mice for 3 wk did not affect the size of the primordial or primary pools, although it did reduce the serum levels of LH and FSH to within the normal range [44]. This suggests that E2 has no direct or indirect effect on the rate of activation of primordial follicles in mice.
Earlier Activation
An earlier onset of follicle activation in ArKO ovaries is supported by the fact that ArKO and estrogen receptor-
(ER) knockout (ERKO) mice undergo precocious follicular development; young E2 deficient mice possess antral follicles before their age-matched wild-type controls [19, 45]. Elevated LH levels, also present in both ArKO and ERKO females, have previously been associated with increased primordial follicle activation [11] and precocious puberty [46]. Although the timing of the initial activation of primordial follicles was not directly assessed in this study, the increased proportion of preantral and antral follicles described previously in 6-wk-old ArKO mice [45] suggests that the onset of primordial follicle activation occurs earlier in development than in wild-type mice. Further experiments are needed to resolve these questions.
Postnatal Germinal Stem Cells
Recently, Johnson and colleagues [1] described the presence of stem cells in the postnatal mouse ovary that could give rise to primordial follicles and thus replenish the follicle pool. We did not examine the mouse ovaries in this study for the presence of these germinal stem cells, which could be influenced by estrogen.
Oocyte and Oocyte Nuclear Diameter
The transition from the primordial to the primary follicle stage in mice is coincident with a change in the shape of the granulosa cells and an increase in oocyte diameter [47]. Similar changes were observed in ArKO ovaries in this study. However, we observed that oocyte and oocyte nuclear diameters in primordial follicles were larger in ArKO mice than in wild-type mice. The reduction in oocyte diameters in ArKO mice after E2 treatment suggests a role of estrogen on oocyte size in primordial follicles. If the effect of estrogen on oocyte diameter is through somatic cell activation/proliferation, then we might have expected to see a coincident increase in primary follicles in E2-treated ArKO animals. It remains unclear which mechanisms E2 uses to influence the diameter of the primordial oocyte and its nucleus. Direct effects imply the presence of ER in the mouse oocyte, which have been described by Wu and colleagues [48]. ArKO ovaries contain both ER and ERß [44], but their cellular localization has not been described. Alternatively, E2 may have indirect effects on the oocyte via ER in the somatic cells [49, 50]. GDF9 is expressed in the oocytes of all growing follicles and absent in oocytes from primordial follicles [51]. Increased oocyte diameters were noted in GDF9-null mice [52], in contrast to ArKO mice, which have increased ovarian GDF9 mRNA expression and increased oocyte diameters. Further work is needed to resolve these differences.
Factors Implicated in Primordial Follicle Formation and Activation
In this study MIS, WT-1, and PCNA proteins were localized to somatic cells of follicles from the primary stage of development onward, as has been described previously in wild-type ovaries [13, 14, 29, 53–55]. However, no differences were detected before or after E2 treatment between ArKO and wild-type mice in immunostaining of these factors in primordial and primary follicles. The differences in immunostaining that were observed were confined to antral follicles, and to the presence of Sertoli and Leydig cells that appear in aged ArKO ovaries. Furthermore, the effects of E2 on the immunostaining patterns were related to improvements in ovarian folliculogenesis and morphology in general. This implies that the factors that we chose to examine do not play a part in the actions of E2 on the primordial and primary pools or on oocyte and oocyte nuclear diameters. This does not preclude a role for these factors in formation and activation of primordial follicles by pathways not involving E2 that have been described previously.
Because ArKO mice have 10-fold elevated serum testosterone levels [42], the possibility that follicle formation and activation are influenced by androgens needs to be considered. While various studies have shown that androgens are important for regulating the number of developing follicles [56–58], no studies to date have directly assessed the effect of androgens on primordial follicle numbers within the ovaries of any species. Despite this, the fact that the androgen receptor is not detectable on primordial or primary follicles ([59–62] and K.L. Britt, unpublished observations) would suggest that androgens do not affect these follicles directly.
MIS, WT-1, and PCNA in the ArKO Ovary
MIS protein staining in ArKO ovaries is consistent with the mRNA expression profiles [44]. Staining was increased in the granulosa cells of early antral and antral follicles of ArKO mice compared with that of wild-type mice at 10 wk of age. MIS was nearly absent by 16 wk. MIS was not observed in the Sertoli cells of ArKO ovaries, although there was distinct localization in the Leydig cells of ArKO ovaries, a site usually reserved for expression of its receptor, not MIS itself [63, 64]. The reduction in MIS staining in 16-wk-old animals reflects the increase in atretic follicles and Sertoli-filled cords, which are not MIS positive. These MIS localization patterns were also observed in one colony of double ER knockout mice [65], but not the other in which Sertoli cells were MIS positive [66].
Within the ovary, WT-1 is expressed in granulosa cells of primary to secondary follicles, and then diminishes with further follicle development [29–31]. WT-1 is also expressed in the nuclei of testicular Sertoli cells of all ages [67]. WT-1 represses the transcription of several genes that are involved in differentiation such as inhibin [30], IGF 1 receptor [68], platelet-derived growth factor, insulin-like growth factor 11, transforming growth factor-ß, and colony-stimulating factor [69], prompting its suggested role in inhibiting the differentiation of immature follicles [30]. It is believed, however, to be involved in follicular survival and growth [4, 70–72].
WT-1 staining was increased in ArKO mice despite the loss of antral follicles [21]. The Sertoli cells in the testicular cord-like structures of adult ArKO ovaries [24] show a high intensity of WT-1 staining. This may explain the elevated levels of DAX-1 observed in ArKO ovaries [44], a nuclear receptor that is dependent on WT-1 for expression [73, 74]. However, the absence of MIS in Sertoli cells of the ArKO ovary suggests that WT-1 expression is not sufficient to maintain MIS expression.
PCNA staining was observed in antral follicles of both ArKO and wild-type ovaries, particularly in the granulosa cells, which is indicative of proliferation, as has been described for antral follicles previously [75]. However, no PCNA staining was detected in either Sertoli or Leydig cells present in ArKO ovaries, indicating a lack of proliferation by these cells. This can be interpreted as showing that they form by transformation from granulosa and interstitial cells, respectively, rather than by the proliferation of Sertoli or Leydig cell precursors. The observation of PCNA staining in primordial follicles that contained both squamous and cuboidal granulosa cells agrees with observations made previously [13, 14, 75] showing that primordial "transitionary" follicles possess proliferating granulosa cells. Furthermore, the expression of PCNA in the squamous granulosa cells of some primordial follicles observed in both wild-type and ArKO mice concurs with the same previous reports [13, 14, 75]. Additionally, granulosa cells within so-called transitory follicles are labeled following by tritiated thymidine (3H-TdR) infusion [76]. Overall, this suggests that granulosa cell proliferation in these primordial follicles precedes their morphological changes.
These data are consistent with E2 facilitating the formation of primordial follicles from germ cell nests, regulating the size of oocytes, and facilitating earlier activation of follicles from the primordial pool. The patterns of staining for expression of MIS, WT-1, PCNA, and the levels of expression of mRNA for GDF9 reflect the changes in follicular differentiation and transformation of ovarian cells to, and the lack of proliferation of, Sertoli and Leydig cells in the ArKO ovary. In conclusion, E2 has a role in controlling the size of the primordial follicle pool and the oocyte size in mice, which may have ramifications for fertility and infertility in mammals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kara Britt, Prince Henry's Institute of Medical Research, Monash Medical Centre Clayton, Block E, Level 4, Clayton, VIC 3168, Australia. FAX: 61 3 9594 6125; kara.britt{at}phimr.monash.edu.au
Received: 4 February 2004.
First decision: 24 February 2004.
Accepted: 9 July 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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