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Endocrinology, doi:10.1210/en.2006-1193
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Endocrinology Vol. 148, No. 4 1707-1716
Copyright © 2007 by The Endocrine Society

Development of Primordial Follicles in the Hamster: Role of Estradiol-17ß

Cheng Wang and Shyamal K. Roy

Departments of Obstetrics and Gynecology (C.W., S.K.R.) and Cellular and Integrative Physiology (S.K.R.), Durham Research Center, University of Nebraska Medical Center, Omaha, Nebraska 68198

Address all correspondence and requests for reprints to: Shyamal K. Roy, Departments of Obstetrics and Gynecology and Cellular and Integrative Physiology, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515. E-mail: skroy{at}unmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 µg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17ß (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 µg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTRADIOL-17ß (E2) STIMULATES granulosa cell proliferation in adult mammals (1); however, its role in the formation and development of primordial follicles remains unclear. It has been proposed that a decrease in serum levels of E2 in newborn rats triggers the formation of primordial follicles, whereas higher levels of E2 near term prevent the process (2). In pregnant mice, plasma E2 levels increase significantly by d 19 of gestation, which is the day of delivery (3). Although light-microscopic morphological studies suggest that ovaries of term fetal mice do not contain primordial follicles, which appear within hours of delivery on d 0 of postnatal life (4, 5), ultrastructural studies indicate that some oocytes are encircled by the cytoplasmic processes of pregranulosa cells resembling primordial follicles on d 18 of gestation (6). In the rat, Montano et al. (7) have demonstrated that although total E2 levels decline by 18% by 4 h after birth, the levels of free E2 actually increase 4-fold by that time. Furthermore, total amounts of E2 decrease significantly in the rat by 48 h after birth, but the percentage of free E2 declines only 1.5-fold. However, similar to mice, the formation of primordial follicles in newborn rats occurs within hours after birth (2, 8). In contrast to rats and mice, morphologically distinct primordial follicles are not visible until 8 d of postnatal life (9, 10) when appreciable amounts of E2 are present in the blood (10, 11). In bovine and sheep fetuses, primordial follicles appear by 90 and 75 d of gestation, respectively (12, 13, 14), when the average concentration of E2 in bovine fetal serum is reported to be 13 pg/ml, whereas the intraovarian concentration is approximately 31 pg/ml (12). Similar data for sheep are not available. In fetal primates, primordial follicles begin to form around the midpoint of gestation (15) when maternal serum E2 levels start to rise from 1 to 2.5–3.0 ng/ml (16). All these lines of evidence suggest that the formation of primordial follicles coincides with comparatively higher levels of E2 in maternal, fetal, or neonatal serum.

Estrogen receptor type 1 (ESR1, also known as ER{alpha}) and ESR2 (also known as ERß) are expressed in fetal human, rat, mouse (1), and hamster ovaries (17). Primordial follicles form in the ovaries of mice null with ESR1 [ER{alpha} knockout ({alpha}ERKO)] (18), ESR2 (ßERKO) (19), or ESR1 plus ESR2 ({alpha}ßERKO) (19) gene, but follicular development at the antral stage is severely compromised (19). However, mouse granulosa cells exclusively express ESR2 that remains functional in {alpha}ERKO mice. Conversely, ESR1 remains functional in ßERKO mice and can cause functional compensation. Although primordial follicles also form in mice null with CYP19A1 gene [aromatase knockout (ArKO)], it should be noted that fetal and neonatal ovaries of CYP19A1+/+ are exposed to maternal E2 (20) because the breeding requires heterozygotes. Finally, a possible role of membrane ESR (21) in somatic cell differentiation in the fetal ovary cannot be overlooked. In fact, ovarian somatic cell differentiation is severely compromised in ArKO mice (20, 22), which can be prevented by E2 replacement for 3 wk (23), suggesting that additional studies are necessary to understand the physiological role of E2 in primordial follicle formation and development. Treatment of pregnant baboons with an aromatase inhibitor throughout the second half of gestation results in a significant attenuation of primordial follicle development even though the oocyte nests and surrounding mesenchymal cells are in place (16). Oocytes in fetal baboons deprived of estrogen have markedly reduced number and size of the microvilli, which are critical for nutrient uptake, and the defect can be restored with a concomitant treatment with E2 (24), suggesting that estrogen action is needed for primordial follicle formation in primates. A long-term treatment of cows with E2 leads to increased development of primordial follicles to primary stage (25). All these lines of evidence suggest that ovarian somatic cells across species have all the components to respond to E2; however, estrogen regulation of perinatal follicular morphogenesis varies across species. The objectives of the present study were to determine whether 1) E2 would play any role in the formation and development of primordial follicles in vivo and in vitro, 2) interruption of E2 action in vivo or in vitro would affect primordial folliculogenesis, and 3) hamster ovaries during perinatal development had the potential to produce E2. We have used golden hamsters to address these objectives because primordial follicles do not appear in the ovary until postnatal d 8 (P8) (10) or until 9 d of culture in vitro (26, 27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult golden male and female hamsters were purchased from Charles River Laboratories (Charles River, MA) and maintained in a climate-controlled room with 14 h light and 10 h dark with free access to food and water according to the Institutional Animal Care and Use Committee (IACUC) and the U.S. Department of Agriculture guidelines. The use of hamsters for this study was approved by the IACUC. Females with at least three consecutive estrous cycles were mated with males on the evening of proestrus, and the presence of sperm in the vagina the next morning was considered d 1 of pregnancy. Hamster gestation lasts for 16 d, and pups are born on d 16 of gestation, which we considered the first day of postnatal life.

E2 cypionate (ECP) was purchased from Pfizer company (Kalamazoo, MI). Phenol red-free DMEM, linolenic acid, BSA for tissue culture, and fine chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). E2 was purchased from Steraloids, Inc. (Newport, RI). ICI 182,780 was purchased from Tocris Bioscience (Bristol, UK). Human holo-transferrin, selenium, and bovine insulin were purchased from Collaborative research (Bedford, MA). Falcon non-tissue-culture-coated inserts and plates, solvents for histology, and other fine chemicals were purchased from Fisher Scientific Co. (Pittsburgh, PA). Plastic embedding medium was from Electron Microscopy Sciences (Hatfield, PA). Antibody to E2 RIA was a generous gift from the late Dr. K. Quadri and optimized in our laboratory (10). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) kit was from Chemicon (Temecula, CA). PCR chemicals were from Roche Molecular Biochemicals (Indianapolis, IN), Pharmacia Biotech Boehringer (Piscataway, NJ), and Invitrogen (Carlsbad, CA).

In vivo effect of E2 and ICI 182,780 on primordial follicle formation and development
Neonatal hamsters were injected sc with 1 or 2 µg ECP in 20 µl sesame oil at 0900 h on P1 or on P1 and P4. ECP was used because it slowly raised serum levels of E2 and maintained it for a long time (28, 29), which was essential to avoid daily injection to pups that would trigger cannibalism by mothers. ECP was injected sc because pups had limited muscle mass, but the route of injection was expected to slow down E2 release further. The dosages were selected from a preliminary dose-response study. The role of endogenous E2 on the formation of primordial follicles was examined by injecting hamsters with 10 µg ICI 182,780 on P1 or on P1 and P4. Control animals received sesame oil vehicle. There were five hamsters in the control and each treatment group. Ovaries were collected between 0900 and 1000 h on P8 and were processed for morphometric evaluation of the formation and development of primordial follicles or detection of apoptosis by TUNEL. Serum from each animal was collected between 0900 and 1000 h on P8 for measuring E2 levels by RIA.

In vitro effect of E2 and ICI 182,780 on primordial folliculogenesis
Ovaries were collected between 0800 and 1000 h from embryonic d 15 (E15) fetal hamsters in phenol red-free DMEM containing antibiotics at room temperature, cleaned of adherent tissues, and cultured in the presence of 0.1 µg/ml insulin, and 1.25 µg/ml transferrin, 1.25 µg/ml selenium, and 10.7 µg/ml linoleic acid (ITS+) as described previously (26, 27, 30) with or without 1, 5, or 10 ng/ml E2 or different dosages of E2 plus 100 nM ICI 182,780 for 9 d. All ovaries were placed in culture by 1200 h. Medium was changed every 48 h. For each group, there were at least three E15 ovaries from separate pregnant hamsters. Ovaries were retrieved at 1200 h on d 9 of culture and processed for morphometric evaluation of the formation and development of primordial follicles or detection of apoptosis by TUNEL.

CYP19A1 mRNA expression during perinatal ovary development and synthesis of E2 in vitro by neonatal hamster ovaries
In the first experiment, ovaries were collected at 0900 h from E13, E15, P2, P4, P6, P9, and P10 hamsters and processed for semiquantitative RT-PCR examination of CYP19A1 mRNA expression. Ovarian RNA from metestrus (d 2) hamsters was used as a positive control. Serum from 15-d pregnant and 2-, 4-, 6-, and 8-d-old neonatal hamsters were collected at 0900 h for measuring serum levels of E2 by RIA. There were three pregnant hamsters for each day of gestation and three neonatal hamsters for each postnatal day.

In the second experiment, eight ovaries from 4-d-old hamsters were collected at 0900 h and cultured for 24 h without or with 5 ng/ml testosterone (T) or T plus 1 ng/ml ovine FSH-20 in 1 ml phenol red-free DMEM with ITS+ at 37 C in 5% CO2 in air. There were eight ovaries for each of the treatment group. P4 ovaries were selected because they were 4 d away from having any primordial follicle but provided adequate tissue mass that could accumulate a detectable amount of E2 in the medium for RIA. The ovaries were used for semiquantitative RT-PCR examination of CYP19A1 mRNA. Because 4-d-old ovaries would be equal to 5-d-old ovaries after 24 h culture, ovarian RNA from 5-d-old hamsters was used for comparison of in vivo and in vitro levels of CYP19A1 mRNA. Ovarian RNA from metestrous (d 2) hamsters was used as positive control. Each group was repeated three times.

Morphometric evaluation of folliculogenesis
Ovaries were fixed in Bouin fixative for 10 h followed by transferring to 50% ethanol for 24 h. Morphometric evaluation was done as described previously (26, 27). Ovaries cultured for 9 d (equal to 8 d of in vivo development) (10) had significant numbers of primordial oocytes (clusters of oocytes without any definite somatic cell partners), and primordial follicles appeared for the first time. Therefore, the total number of oocytes with a nucleolus, regardless of their follicular association, was counted in a given optical field. Next, follicles at various stages of development corresponding to those oocytes were counted. The fields were chosen at random from the entire ovary, and 300 oocytes for each ovary were counted. The proportion of follicles relative to the oocytes was calculated. An oocyte surrounded by at least one flattened parenchymal cell was considered a primordial follicle, whereas a primary follicle was defined as one surrounded by a complete layer of mostly cuboidal granulosa cells. Early secondary follicles contained more than one layer of cuboidal granulosa cells. No attempt was made to classify follicles in subgroups. The average diameter of the oocytes was calculated from two perpendicular diameters using a calibrated eyepiece micrometer.

Small pieces of uterus were collected during ovary retrieval, cleaned of adherent tissue, fixed overnight in Bouin’s fixative, and embedded in JB4 plastic using a protocol similar to that used for ovaries (27). Care was taken to keep the uterus vertical during embedding, and 5-µm sections were processed for routine hematoxylin and eosin (H&E) staining. Digital images were captured under a x10 objective using a Leica DEI300 color camera and Openlab image analysis software. All images containing a 10-µm size marker were cropped to the same size using Adobe PhotoShop CS2 software to fit in one image plate. Because uterine morphology changed under different treatment conditions and sections were oriented at random on slides, some images showed more stroma. However, emphasis was given to show epithelial layer as well as considerable stroma for each group for comparison. The average uterine diameter in micrometers was determined from two perpendicular measurements of several sections for each animal. The average thickness of the epithelial cell layer was determined from multiple longitudinal measurements along the layer from the base of the cell to the apical surface of several sections for each animal. The average number of cells in epithelial and stromal layers was determined by counting nuclei present in nonoverlapping 30-µm squares placed randomly over several sections for each animal. Each group had uteri from more than three animals to obtain a mean ± SEM.

RIA of E2
E2 levels in the serum and culture medium were measured by a specific RIA as described previously (31, 32). The assay measured total E2, and the cross-reactivity of the antibody with T was 0.004%. The sensitivity of the assay was 2 pg/tube. All samples were assayed in one time to avoid interassay variation. Intraassay variation was less than 5%.

Detection of apoptosis by TUNEL
Apoptosis of ovarian cells was detected by ApopTag fluorescein in situ apoptosis detection kit. The 7-µm-thick ovary sections were fixed with 1% paraformaldehyde and used for TUNEL detection of fragmented DNA. Nuclei were stained with propidium iodide. The number of cells showing green fluorescence, regardless of the type or stage of apoptosis, were counted in each section. This approach of quantification of apoptotic cells was necessary because the higher sensitivity of TUNEL fluorescence signal was capable of identifying cells at a very early stage of apoptosis that could not be identified by routine H&E staining (chromatopyknosis). Therefore, the presence of pyknotic nuclei in H&E-stained sections varied across different treatment groups. There were three ovaries in each group.

RT-PCR analysis of CYP19A1 mRNA expression in ovarian RNA
Total RNA was extracted with Trizol and RNeasy kit (QIAGEN, Inc., Valencia, CA) and quantified using Ribogreen RNA quantitation kit (Invitrogen) as described earlier (27). Ovarian RNA of metestrous hamsters was used as positive control. One microgram RNA was reverse transcribed and amplified by PCR as described earlier (27, 33, 34, 35). The primer sets were as follows: for CYP19A1, forward 5'-GGACTTGAAAGACGAGATCG-3' and reverse 5'-AGCCTGTGCATTCTTCCGAT-3'; and for actin, forward 5'-GGGCCAGAAGGACTCGTACG-3' and reverse 5'-CACAGCTTCTCTTTGATGTCACGCAC-3'. Actin mRNA in 20x diluted cDNA samples was amplified to validate the specificity of CYP19A1 mRNA expression because actin mRNA levels remained stable in the hamster ovary during development (17). In initial experiments, the identity of PCR products was verified by sequencing. PCR-amplified products were fractionated in 1% agarose gel containing ethidium bromide and digitized in a UVP (Upland, CA) gel documentation instrument. CYP19A1 mRNA levels were presented as OD relative to that of actin mRNA.

Statistical analysis
All cultures and immunofluorescence localizations were repeated at least three times using ovaries from different fetuses. Ovaries from untreated and E2- or ICI-treated groups were cultured in parallel. Ovaries from E15 embryos of each pregnant hamster were pooled to obtain one sample for each embryonic age. There were three pregnant hamsters for each embryonic age to obtain an n = 3. RNA sample for each postnatal day was prepared from ovaries pooled from three postnatal hamsters, and there were three samples for each day. Serum levels of E2 were detected from five samples each using specific RIA (36). All quantitative data were analyzed by one-way ANOVA with Scheffé’s post hoc test using StatView software (SAS Institute, Inc., Cary, NC). The level of significance was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo effect of E2 on follicular morphogenesis
The objectives were to determine whether exogenous administration of E2 would stimulate the formation of primordial follicles and their development into subsequent stages. The rationale of selecting P1 and P4 for ECP or ICI 182,780 injection were 1) to raise serum E2 levels above the baseline or to prevent the endogenous E2 from acting (37) long before the morphologically defined primordial follicles appeared in the ovary on P8 (10) and 2) to sustain the E2 or ICI 182,780 levels during the critical period of follicular morphogenesis. Because inadequate levels of serum E2 or a lack of E2 action in general could present negative results for the ovary, serum levels of E2 and its effect on a known target, such as uterus, were determined. Serum E2 levels on P8 were 100 pg/ml after a single injection of 1 µg ECP on P1. The levels were at approximately 200 pg/ml when the injection was given on P4 and did not increase further with injection on P1 and P4 (Fig. 1Go). Doubling the dose to 2 µg with injection on P1 and P4 raised serum levels of E2 to 400 pg/ml (Fig. 1Go). These results seem to indicate that the half-life of ECP in serum is 4 d when administered sc.


Figure 1
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  		FIG. 1. Serum levels of E2 in P8 hamsters after a sc injection of 1 µg ECP on P1, P4, or on both days or a sc injection of 2 µg ECP on P1 and P4. Each bar represents a mean ± SEM. P < 0.05 for bars with different letters; P > 0.05 for bars with the same letter.

 
Compared with vehicle-treated animals, either dose or treatment regimen of ECP significantly increased uterine diameter (Fig. 2AGo), thickness of the epithelial layer (Figs. 2BGo and 3Go, A–C), and number of epithelial cells per unit area (Figs. 2CGo and 3Go, A–C) by P8. In contrast, the number of stromal cells per unit area decreased significantly in pups treated for 2 d with 1 or 2 µg ECP (Figs. 2CGo and 3Go, A–C). Treatment with ICI 182,780 caused a small but significant reduction of uterine diameter (Fig. 2AGo) and number of epithelial cells (Figs. 2CGo and 3DGo) but did not affect the thickness of the epithelium or the number of stromal cells compared with the vehicle-treated animals (Figs. 2Go, B and C, and 3D).


Figure 2
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  		FIG. 2. Diameters (A), epithelial thickness (B), and the number of epithelial and stromal cells (C) of the uteri of P8 hamsters injected sc with 1 or 2 µg ECP on P1 or P1 and P4 or 10 µg ICI 182,780 on P1 and P4. Methods of measurement of uterine diameter, epithelial thickness, and cell number have been discussed in Materials and Methods. Each bar represents a mean ± SEM. P < 0.05 for bars with different letters; P > 0.05 for bars with the same letter.

 

Figure 3
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  		FIG. 3. Morphology of the uteri of P8 hamsters treated with ECP or ICI 182,780 as described in Fig. 2Go. A, Vehicle; B, 1 µg ECP on P1; C, 1 µg ECP on P1 and P4; D, 10 µg ICI 182,780 on P1 and P4. Note epithelial hypertrophy and hyperplasia after ECP treatment.

 
Under endogenous endocrine milieu, around 10% of the oocytes became primordial follicles by P8 (Figs. 4Go and 5Go). Distinct oocyte clusters surrounded by undifferentiated somatic cells were present throughout the ovary on P8, and many of the oocytes in the clusters were in pachytene stage (arrowhead) of the first meiotic prophase [compare the chromosomal arrangement in the oocyte of primordial follicles (S0) with those in the cluster, Fig. 4Go]. The percentage of primordial follicles increased slightly but significantly in animals treated with 1 µg E2 on P1; however, most notably, many of them moved to the primary stage, which did not occur in vehicle-treated animals (Fig. 5AGo). The percentage of primordial and primary follicles increased markedly in animals treated with 1 µg E2 on P1 and P4. Furthermore, well-developed primary follicles (S1) and a few secondary follicles (S2) were visible (Fig. 4Go). Because the proportion of secondary follicles was low, the information was not included in Fig. 5AGo. Compared with vehicle, 2 µg E2 for 2 d also stimulated the formation of primordial follicles and their development into primary stage, but the increase was significantly lower than that obtained with the 1-µg dose for 2 d (Fig. 5AGo). Treatment with ICI for 1 or 2 d resulted in a small but significant reduction in primordial follicle formation (Fig. 5AGo). The overall morphology of ICI 182,780-treated ovaries was similar to those in untreated P8 animals, except they contained numerous pyknotic cells (darkly stained nuclei; Figs. 4Go and 5AGo).


Figure 4
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  		FIG. 4. Morphology of P8 ovaries exposed in vivo to vehicle (A), 1 µg ECP on P1 and P4 (B), or 10 µg ICI 182,780 on P1 and P4 (C). GC, Granulosa cells; OC, oocyte cluster; S0, primordial follicle; S1, primary follicle; S2, secondary follicles with two layers of granulosa cells. Note the presence of pachytene chromosome in the oocytes in the cluster (arrowhead, compare with the oocyte of S0 follicle), primary follicles with distinct cuboidal granulosa cells, and multilayer follicles in ECP-treated ovaries and a lack of follicle development with appreciable pyknotic cells in ICI 182,780-exposed ovaries. Bars, 10 µm.

 

Figure 5
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  		FIG. 5. Morphometric quantification of primordial and primary follicle formation (A) and quantification of apoptotic cells by TUNEL (B) in P8 ovaries after in vivo treatment with 1 µg ECP or 10 µg ICI 182,780 on P1 or P1 and P4 or 2 µg ECP on P1 and P4. The proportion of follicles was determined as a percentage of total number of oocytes counted, and the number of apoptotic cells was expressed per section. Each bar represents a mean ± SEM. P < 0.05 for bars with different letters; P > 0.05 for bars with the same letter.

 
An increase in primordial follicle number could also be due to reduction in apoptosis that was an integral phenomenon of ovarian morphogenesis during perinatal development (38). Because nuclear apoptosis at an early stage was difficult to identify with routine H&E staining, we used fluorescence-based TUNEL to detect apoptotic cells regardless of their type and stage of apoptosis. Consistent with our previous findings (27), TUNEL-positive cells were also present in ovaries of untreated normal animals (Fig. 5BGo), but virtually all apoptotic cells were oocytes (data not shown). Interestingly, E2 at both dose and time levels significantly suppressed apoptosis (Fig. 5BGo). Paralleling the decrease in primordial follicle number, the number of apoptotic cells increased sharply with ICI 182,780 treatment (Fig. 5BGo).

In vitro effect of E2 on follicular morphogenesis
The rationale was to examine whether the in vivo effect of E2 on primordial folliculogenesis could be reproduced in vitro for establishing a direct role of E2 on ovarian cells. Ovaries contained several clusters of oocytes surrounded by somatic cells (Fig. 6AGo). Consistent with our previous findings, approximately 9% of oocytes became primordial follicles by d 9 of culture (Fig. 7AGo), and ovaries contained a basal number of apoptotic cells (Fig. 7BGo). Furthermore, most of the apoptotic cells appeared to be in the early stage because they were difficult to identify in H&E-stained sections (Fig. 6AGo). Exposure to 1 ng/ml E2 markedly enhanced primordial follicle formation and stimulated their development to primary stage (Figs. 6BGo and 7AGo). Numerous primary follicles with a distinct layer of cuboidal granulosa cells were visible (Fig. 6BGo); however, the number of apoptotic cells was not significantly different from the untreated group (Fig. 7BGo). Increasing the E2 dose to 5 ng/ml (Fig. 6CGo) or 10 ng/ml (data not shown) resulted in the appearance of somatic cells with pyknotic nuclei and strong eosin-stained cells without nuclei (ghost cells; Fig. 6CGo). These higher doses not only failed to stimulate the formation of primordial follicles (Fig. 7AGo) but also significantly increased the number of apoptotic cells (Fig. 7BGo) compared with the untreated group. ICI 182,780 completely blocked the stimulatory effect of 1 ng/ml E2 on primordial follicle formation and development and caused a considerable appearance of ghost cells (Figs. 6DGo and 7AGo). However, there was no significant increase in TUNEL-positive cells (Fig. 7BGo), probably due to the presence of cells without nuclei. Although ICI 182,780 treatment failed to reverse the effect of higher doses of E2 on primordial follicle formation (Fig. 7AGo), it significantly attenuated their effect on apoptosis (Figs. 6EGo and 7BGo).


Figure 6
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  		FIG. 6. Morphology of E15 ovaries cultured for 9 d in vitro in the absence or presence of E2 with or without ICI 182,780. Ovaries were cultured with 0.01% ethanol (solvent for E2) (A), 1 ng/ml E2 (B), 5 ng/ml E2 (C), 1 ng/ml E2 plus 100 nM ICI 182,780 (D), or 5 ng/ml E2 plus 100 nM ICI 182,780 (E). IC, Interstitial cells; O, oocyte; OC, oocyte cluster; S0, primordial follicle; S1, primary follicle; arrowheads, apoptotic cells. Note the presence of discrete eosin-stained ghost cells when the ovaries were exposed to 5 ng/ml E2 or the effect of 1 ng/ml E2 was blocked by ICI 182,780. Bars, 10 µm.

 

Figure 7
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  		FIG. 7. Morphometric quantification of primordial and primary follicle formation (A) and quantification of apoptotic cells by TUNEL (B) in E15 ovaries cultured for 9 d in vitro in the absence or presence of 1, 5, or 10 ng/ml E2 with or without 100 nM ICI 182,780. The proportion of follicles was determined as a percentage of total number of oocytes counted, and the number of apoptotic cells was expressed per section. Each bar represents a mean ± SEM. P < 0.05 for bars with different letters; P > 0.05 for bars with the same letter.

 
Expression of CYP19A1 in the fetal and newborn hamster ovaries and production of E2 in vivo and in vitro
Whereas administration of E2 in vivo or in vitro could induce primordial follicle formation and development, key questions were whether 1) neonatal ovaries were exposed to endogenous E2 during the formation of primordial follicles, 2) neonatal ovaries had the ability to produce estrogen, and 3) FSH would modulate ovarian E2 synthesis. Maternal serum levels of E2 were relatively high on d 15 of gestation (Fig. 8AGo). Because of technical difficulties, no attempt was made to measure blood levels of E2 in E15 embryos. Appreciable levels of E2 were present in P2 pup serum (Fig. 8AGo). Serum levels of E2 declined significantly by 4 d of age followed by increases through P8 (Fig. 8AGo). It is noteworthy that increase in serum levels of E2 on P8 corresponded to the first appearance of primordial follicles in the ovary (10).


Figure 8
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  		FIG. 8. Ability of neonatal hamster ovaries to synthesize E2. A, Serum levels of E2 in pregnant hamsters on d 15 of gestation and in neonatal female hamsters during the formation of primordial follicles; B, semiquantitative RT-PCR determination of CYP19A1 and actin mRNA expression in hamster ovaries from fetal through P10 when primordial follicles first appeared in the ovary; top, ethidium-bromide-stained cDNA; bottom, OD of amplified CYP19A1 cDNA normalized against actin cDNA. Ovarian RNA from diestrous hamsters was used as positive control for CYP19A1 mRNA. C, In vitro synthesis of E2 from T by P4 ovaries. Eight ovaries were cultured for 24 h with 0.01% ethanol (vehicle), 1 ng/ml ovine-FSH-20, 5 ng/ml T, or T plus FSH, and E2 accumulated in the medium was quantified by RIA. Values obtained for medium with T but without ovaries (blank) were subtracted from those of media with ovaries to determine true values for E2. D, Semiquantitative RT-PCR determination of CYP19A1 and actin mRNA expression in P4 ovaries cultured for 24 h with 0.01% (vehicle), 1 ng/ml ovine-FSH-20, 5 ng/ml T or T plus FSH; top, ethidium-bromide-stained cDNA; bottom, OD of CYP19A1 cDNA normalized against actin cDNA. Ovarian RNA from diestrous hamsters was used as positive control for CYP19A1 mRNA. Each bar represents a mean ± SEM. P < 0.05 for bars with different letters; P > 0.05 for bars with the same letter.

 
Distinct CYP19A1 mRNA was present in E13 ovarian RNA (Fig. 8BGo). Expression of CYP19A1 mRNA in neonatal ovaries increased steadily from P4 through P10, corresponding to serum levels of E2 (Fig. 8BGo). No change in ovarian actin level was noted (Fig. 8BGo), indicating the specificity of CYP19A1 gene expression during neonatal ovary development.

In the absence of exogenous T, P4 ovaries could make very low levels of E2 with or without 1 ng/ml FSH (Fig. 8CGo). Ovarian production of E2 increased significantly after the administration of T, and the rate of synthesis increased further when FSH was administered together with T (Fig. 8CGo), suggesting that CYP19A1 mRNA was translated into functional aromatase enzyme during neonatal ovary development.

To determine whether 24 h culture with T and/or FSH would result in an increased CYP19A1 mRNA expression in vitro, levels of CYP19A1 mRNA were examined. The values were compared with P5 ovaries because P4 ovaries would be technically P5 ovaries after 24 h culture. In the absence of T or FSH support, ovarian CYP19A1 mRNA expression declined markedly compared with P5 ovaries developed in vivo (Fig. 8DGo). Either T or FSH maintained the expression of CYP19A1 mRNA similar to in vivo condition, but FSH significantly up-regulated the expression as well (Fig. 8DGo). However, CYP19A1 mRNA expression increased markedly in ovaries cultured with T plus FSH (Fig. 8DGo), suggesting that FSH regulation of CYP19A1 mRNA expression in neonatal ovaries requires estrogen support.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study provide a direct evidence that E2 plays a critical role in somatic cell differentiation into pregranulosa cells leading to the formation and development of primordial follicles. Furthermore, it is apparent that E2 has dual effects on oocyte and somatic cell communication depending on the dosage. It can be speculated that whereas E2 at relatively lower levels supports somatic cell and oocyte interaction during the critical phase of cell assembly, at higher levels it adversely affects the process; hence, cellular degeneration and suppression of primordial follicle formation occur. This is further evident from the marked increase in apoptotic cells in vitro by higher doses of E2 and its reversal by ICI 182,780. However, cellular commitment that is already established during fetal development in vivo under endogenous E2 can no longer be disrupted by higher doses of E2; hence, a basal number of primordial follicles form regardless of treatment. Although 1 ng/ml E2 in vitro seems to be more than the levels present in vivo, follicular development is not compromised, suggesting the presence of a threshold of E2 levels above which the detrimental effect occurs. This is evident when serum levels of E2 are raised to 400 pg/ml. It can be speculated that free E2 in the culture medium is in the low picogram range because BSA present in the culture medium is expected to bind a significant amount of E2 and lower the concentration to an effective biological level. The exact amount of free E2 in the medium is unknown because the assay measures total E2 concentration. The small but significant effect of ICI 182,780 on primordial follicle formation provides evidence that endogenous levels of E2 during early postnatal development support cellular differentiation necessary for the formation of primordial follicles. It has been reported that an effective reduction of endogenous E2 effect requires daily injection of a large dose of ICI 182,780 (37), which may explain the less than robust decline in primordial follicle formation by ICI administered on P1 and P4. The rationale for not using ICI injection daily or at higher doses is to avoid any toxic effect in neonatal hamsters and cannibalism by the mother, which is a common phenomenon in the hamster when pups are handled frequently. The marked increase in uterine epithelial cell number and size by P8 in response to ECP in vivo provides evidence that a lower dose of steroid can maintain an effective concentration of E2 in serum for biological activity. A significant decrease in stromal cell number after 2 d of ECP treatment may indicate E2-induced stromal edema. Similarly, a decrease in uterine diameter and the number of stromal cells without affecting the thickness of the epithelial cell layer after ICI 182,780 treatment in vivo suggests a block in endogenous E2 action on uterine cells. The presence of uterine hypoplasia in {alpha}ERKO mice, and a further 2-fold reduction in uterine diameter and wall thickness associated with decreased number of cells in {alpha}ßERKO mice have been reported (19). Because ICI 182,780 is expected to block the action of both types of classic ESR, the present findings on uterine phenotype resemble {alpha}ßERKO more than {alpha}ERKO. In fact, the thickness of uterine epithelium is severely reduced in {alpha}ßERKO mice compared with those in the {alpha}ERKO (19). Because the study focuses on primordial follicle formation, extensive analysis of the effect of E2 or ICI 182,780 on uterine cells has been avoided.

The results of E2 induction of primordial follicle formation and development in the hamster ovary are consistent with those reported in the baboon (16, 24) and cow (25). In contrast to these findings, Kezele and Skinner (2) have proposed that E2 action is detrimental to primordial follicle formation in the rat. However, it should be noted that the authors raise the serum E2 concentration to 544.7 ng/ml with a daily injection of 3.3 µg E2 for 4 d to newborn rats (2). We demonstrate that raising the serum E2 levels from 200 pg/ml to 400 pg/ml attenuates the formation and development of primordial follicles; therefore, the positive or negative effect of E2 on primordial follicle formation and development appears to depend on the dose of the steroid. In the human fetus, the number of apoptotic oocytes starts to decrease from 22 wk of gestation (39), correlating with an increase in maternal (40) and fetal (41) E2 levels, and becomes lowest near term (39). Based on our findings, it is very clear that E2 concentration above 1 ng/ml in vitro adversely affect folliculogenesis, and endogenous E2 levels are at the low picogram level. Therefore, picogram per milliliter levels of E2 present in vivo during normal neonatal ovary development are in fact essential for somatic cell survival and primordial follicle formation. This is further supported by the effect of ICI 182,780. We can conjecture from the results that E2 at low levels not only supports somatic cell survival but also stimulates cells to differentiate. If E2 support is compromised by a block in E2 action or lack of E2 during the critical process of cellular differentiation, affected cells become apoptotic. During 9 d of culture, these cells eventually lose their nuclei and become eosin-stained ghost cells. The lack of nuclei may explain the unaltered number of TUNEL-positive cells in ovaries exposed to 1 ng E2 plus ICI 182,780 despite the histological evidence of cellular degeneration. Significant decreases in the number of primordial follicles and a close to significant reduction in the number of primary follicles have been observed in aromatase-deficient (ArKO) mice (42). However, E2 replacement from wk 7–10 fails to alter this phenotype (42). Because the study includes adult wild-type and ArKO mice, it is unclear whether the lack of E2 has caused a reduction in primordial follicle formation or an increase in attrition, but the results establish a supportive role E2 in mice. Our data together with those reported by Vomachka and Greenwald (11) indicate that serum levels of total E2 in neonatal female hamsters do not exceed 40 pg/ml, but the present results also demonstrate that raising serum E2 levels to as much as 180 pg/ml by ECP results in the formation and development of primordial follicles. It can be speculated that an upper level set point may determine the positive or negative effect of E2 on the formation and development of primordial follicles. Conversely, E2-binding proteins, such as albumin (in vivo and in vitro) or {alpha}-fetoprotein (in vivo) (43) may play an important role in maintaining free E2 levels within physiological limits. Treatment of newborn rats with 10 µg estradiol benzoate per day for 5 d results in a lower number of multilayer follicles as early as 6 d of postnatal life (44).

We have demonstrated that ESR1 and ESR2 mRNA and protein are expressed in hamster ovaries during neonatal development, and the expression is regulated by FSH (17). The presence of ESR in fetal and neonatal ovaries has also been reported for many species (1) including baboon (45). The presence of CYP19A1 mRNA and production of E2 from T in vitro by P4 hamster ovaries provide strong evidence that functional aromatase enzyme exists in neonatal hamster ovaries. Furthermore, the expression of CYP19A1 mRNA appears to be maintained by E2 and regulated by FSH; however, some direct role of androgen cannot be ruled out. Luo and Wiltbank (46) have shown that either E2 or T can stimulate CYP19A1 mRNA expression in bovine granulosa cells, and the effect is blocked by ICI 182,780. In contrast, T or the product E2 seems to only maintain CYP19A1 mRNA levels in neonatal hamster ovaries, whereas FSH up-regulates CYP19A1 mRNA expression. The increase in E2 production or CYP19A1 mRNA expression in the presence of FSH and T in vitro may suggest an increased expression of functional FSH receptors; however, future studies will address this issue. T or dihydrotestosterone can augment FSH receptor mRNA expression in bovine granulosa cells in vitro (46).

In summary, the results of the present study provide strong evidence that functional CYP19A1 enzyme is present in neonatal hamster ovary, which can produce E2 both in vivo and in vitro. E2 plays an important role in ovarian somatic cell differentiation into pregranulosa cells and helps in oocyte-granulosa cell communication resulting in the formation and development of primordial follicles. The present data along with our previous findings (10) suggest strongly that at least part of the FSH regulation of primordial follicle formation is mediated by E2.


   Footnotes
 
This work was supported by a grant (R01-HD38468) from the National Institute of Child Health and Human Development, National Institutes of Health, to S.K.R. C.W. was supported by a postdoctoral fellowship from the Lalor Foundation, USA.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 28, 2006

Abbreviations: ArKO, Aromatase knockout; E2, estradiol-17ß; E15, embryonic d 15; ECP, estradiol cypionate; ESR, estrogen receptor; ERKO, estrogen receptor knockout; H&E, hematoxylin and eosin; P8, postnatal d 8; T, testosterone; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Received August 30, 2006.

Accepted for publication December 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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