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Prince Henry’s Institute of Medical Research and Department of Anatomy, Monash University (N.G.W.), Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Prof. J. K. Findlay, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: jock.findlay{at}med.monash.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß, both of which are present in granulosa cells of mice and
rats (4, 5).
A requirement of folliculogenesis for E2 has been
implied in earlier studies (reviewed in Refs. 2, 6) and more
recently in murine models with targeted disruption of the genes for
either ER (ERKO) or ERß (ßERKO) (7, 8). However, neither of
these models is completely free of the capacity to transduce the
E2 signal, given that the alternative ER subtype
remains. These models allow neither definition of the critical period
of nor of the absolute requirement for E2 action
during folliculogenesis.
The recent development of a mouse model lacking the capacity to produce estrogen due to targeted disruption of the Cyp 19 (aromatase) gene (9) provides an opportunity to define the role of E2 in ovarian function. The phenotype of the aromatase knockout (ArKO) female mouse at 12–14 weeks of age was described in a preliminary study (9) as infertile, with ovaries lacking corpora lutea (CL).
The present studies report on the ovarian phenotype of wild-type, heterozygous, and ArKO mice at 10–12 and 21–23 weeks and 1 yr of age. The aim was to determine the impact of aromatase deficiency on the ovarian morphology and the number of follicles that grow to the primary, secondary, and antral stages of development. In addition, the gonadotropin levels within which the ovarian phenotypes are expressed were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sample collections
Vaginal smears were performed daily in the mice. ArKO mice
do not exhibit an estrous phase, but display vaginal smears reminiscent
morphologically of diestrus and proestrus, alternating between these
phases but without any regular cyclicity. Animals in groups of three or
four were killed at a stage of the estrous cycle other than estrus for
controls and heterozygotes or at random for the ArKO females. Blood was
obtained by cardiac puncture and was allowed to clot; serum was
separated and stored at -20 C for gonadotropin RIA. Uterine weight was
recorded as an index of estrogenicity. One ovary from each animal was
frozen in OCT compound (Tissue-Tek, Miles, Elkhart, IN), and stored at
-80 C. The other ovary was fixed in formalin and embedded in paraffin
blocks, which were serially sectioned at 7 µm. Every fourth section
was stained using a modified Masson’s Trichrome (10) technique,
dehydrated in ethanol, and coverslipped using DPX (BDH Laboratory
Supplies, Poole, UK). Masson’s Trichrome stains collagen fibers blue;
cytoplasm red, and nuclei blue-black.
Morphological classification of growing follicles
Follicle types in ovarian cross-sections were defined as
follows. Primary follicles comprised an oocyte surrounded by a single
layer of cuboidal granulosa cells. Secondary follicles comprised an
oocyte surrounded by two or more layers of granulosa cells with no
antrum. Antral follicles were distinguished by an antrum within the
granulosa cell layers enclosing the oocyte. An extensive analysis of
primordial follicles in ArKO ovaries is currently underway, and thus,
primordial follicle data will not be reported here. Follicles were
determined to be atretic if they displayed two or more of the following
criteria within a single cross-section: more than two pyknotic nuclei,
granulosa cells within the antral cavity, granulosa cells pulling away
from the basement membrane, and/or uneven layers of granulosa
cells.
Stereological analysis
Stereological assessment of numbers of growing follicles
(primary, secondary, and antral) was performed using a modification of
the contemporary fractionator method (11). This approach involves
counting all oocyte nuclei in a known fraction of the ovary. The total
number of oocytes per ovary, and therefore follicle number, was then
estimated by multiplying the number counted by the inverse of the
sampling fractions. In this instance, every fourth section was mounted
for stereological examination. Sections were sampled using a systematic
uniform random sampling scheme (12) generated by a computer-driven
stage (Multicontrol 2000, ITK, Lahnau, Germany) mounted on an
Olympus Corp. BX50 microscope (Albertslund,
Denmark). The microscope image was captured using a Pulinex
TMC-6 (Sunnyvale, CA) video camera and interfaced to the counting
frames that were generated using CASTGRID (version 1.10) software,
supplied by Olympus Corp. In brief, the boundary of the
sections was mapped at medium power (x20). The software was then used
to select fields for counting starting in the upper lefthand corner of
the section and advancing by 300 µm in the X direction until the
section was no longer seen. The stage was then moved 300 µm in the Y
direction, and the process was repeated until the whole section was
sampled. A sampling frame consisting of two inclusion and two exclusion
boundaries, as defined by Gundersen (13), was used. Some nuclei gave
rise to profiles in adjacent sections, resulting in an overestimate of
the number of oocytes, which was corrected using Abercrombie’s method
(14) with the addition of the lost cap correction described by Floderus
(15). Both nuclear and follicle diameters associated with counted
oocyte nuclei were measured. Profiles of oocyte nuclei and follicles
were close to circular. Slight deviations from the circular were
accommodated by calculating the geometric mean of the short and long
axes. The mean diameter of nuclei used for the purposes of the
Abercrombie correction was the mean of the upper 30% of the diameters
measured.
Numbers of CL
An estimate of the total number of CL per ovary was made using a
physical dissector approach (16). As CLs had a diameter greater than
175 µm, every 25th section was projected onto paper, and all CLs were
traced. Traces were then compared to determine the total number of CLs
per ovary.
Terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end
labeling (TUNEL) assay
Reagents supplied by Roche Molecular Biochemicals
(Mannheim, Germany) were used for TUNEL staining. Selected
formalin-fixed paraffin sections cut at 7 µm were dewaxed in
histolene, rehydrated with ethanol, and washed in 0.01 M
PBS. Proteinase K (15 µg/ml) was applied to the sections for 15 min
at 37 C, followed by washing in PBS. At this point one section per
group was subjected to deoxyribonuclease treatment (0.375 U/µl) for
15 min at 37 C (positive control). A cocktail of dioxygenin-DNA
labeling mix, terminal transferase, and cobalt chloride, prepared in
1 x terminal transferase buffer was applied to the slides and
incubated at 37 C for 1 h. After washing the slides in PBS, the
slides were blocked in buffer 2 (blocking reagent dissolved in 0.1
M maleic acid and 0.15 M NaCl) for 30 min
before the addition of an alkaline phosphatase-conjugated sheep
antidioxygenin (1:2000), to the sections. After a 1-h incubation
at room temperature, the antibody was washed off with three changes of
PBS, each for 10 min. An enzyme-catalyzed color reaction with nitro
blue tetrazolium chloride (337.5 µg/ml),
x-phosphate/5-bromo-4-chloro-3-indolyl-phosphate (175
µg/ml), and 0.72 mg/ml levamisole (a blocker of endogenous alkaline
phosphatase) produced an insoluble precipitate visualizing hybrid
molecules in the apoptotic cells. The color reaction was left to
develop in the dark for 30 min, at which time it was stopped by washing
the sections with distilled water. After a 30-min incubation in 95%
ethanol, sections were dehydrated in 100% ethanol (twice, 3 min each
time) and cleared in Histosol (twice, 3 min each time) before being
coverslipped with DPX.
Gonadotropin assays
LH and FSH were measured in specific RIAs using reagents
supplied by the NIADDK (LH antiserum S-10, FSH antiserum S-11) with rat
standards (rLH RP-2, rFSH RP-2) and tracer. All samples for each
hormone were measured in a single assay, with intraassay coefficients
of variation of 6.5% and 6.9% for FSH and LH, respectively. The
sensitivities of the assays were 1.15 ng/ml and 96 pg/ml for FSH and
LH, respectively (at the 90% effective dose).
Nonspecific esterase histochemistry
To establish whether cells infiltrating the ovary were
macrophages, the sections were stained for nonspecific esterase, an
enzyme prevalent in monocytes and macrophages (17). Representative
sections from three or four ovaries of ArKO and wild-type animals at
10–12 weeks and 1 yr were selected for esterase staining. Frozen
sections were fixed for 30 min in 4% paraformaldehyde, followed by two
washes in 0.15 M
Na2HPO4 buffer (pH 7.5).
Immediately before use, 1 ml 4% NaNO2 was added
to 1 ml 4% pararosaniline HCl, and 1.6 ml of this mixture were then
combined with 0.5 ml 1% napthyl acetate and 20 ml 0.15 M
Na2HPO4 buffer (pH 7.5) and
mixed thoroughly. The solution was added dropwise to the sections and
incubated for 20–30 min at room temperature. The slides were then
washed in three changes of distilled water, dehydrated in ethanol, and
coverslipped with DPX.
Statistical analyses
Data are presented as the mean ± SEM.
Statistical analysis was performed using SigmaStat statistical software
(version 2.0, Jandel Corp., San Rafael, CA). Longitudinal comparisons
of data at 10–12 weeks, 21–23 weeks, and 1 yr of age and comparisons
within each age group across genotypes were performed using an ANOVA in
conjunction with a Tukey test. In some cases it was necessary to
normalize the data by log transformation before analysis. If normality
or equal variance failed, a Kruskal-Wallis test in conjunction with
Dunn’s multiple comparisons test was performed.
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Results |
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). By 1 yr of age, the uterine weights
of the ArKO mice were only 30% of those recorded for wild-type and
heterozygous groups, both of which were similar.
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). At 10–12 weeks of age, the
heterozygotes exhibited significantly higher levels of FSH than the
wild-type animals, although by 21–23 weeks of age, the heterozygotes
had FSH levels similar to those of the wild-type animals. By 1 yr of
age, the levels of FSH in wild-type animals had increased 2-fold
relative to those in 10- to 12-week wild-type animals. FSH levels in
the ArKO mice were 3-fold higher than levels measured in the serum
collected from 1-yr-old wild-type mice. Interestingly, FSH levels in
heterozygotes were only 50% of wild-type levels at 1 yr (Fig. 1).
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). At 21–23 weeks of age, LH
levels in heterozygous and wild-type animals were similar. By 1 yr of
age, LH levels in ArKO mice were approximately 3-fold higher than those
in the serum of wild-type and heterozygous mice (Fig. 1).
Morphological description of ovaries
Wild-type ovaries at all ages studied contained follicles at
all stages of development and CL (Fig. 2, A and B, and Table 2). At 21–23 weeks
and 1 yr of age, some antral follicles of wild-type ovaries showed
disruption of the cellular layers and the presence of pyknotic nuclei,
typical of apoptotic granulosa cells. Healthy follicles were found
adjacent to atretic follicles. The cellular architecture of the ovary,
particularly that of the interstitial region, was well defined and
compact, as shown in the example at 10–12 weeks of age (Figs. 2, A and
B, and 4F).
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shows a representative section
of a 10- to 12-week-old heterozygous ovary. Open oval structures,
postulated to be the remains of early stage follicles, were present
within the interstitial region of the heterozygous ovaries (Figs. 2, C
and D, and 4I). These structures in most cases possessed a layer of
squamous and/or cuboidal granulosa cell-like cells around the periphery
(Figs. 2D and 4I). Pyknotic nuclei and cellular debris were present in
the antra of some large follicles, particularly at 1 yr (data not
shown).
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and Table 2). Many of the antral
follicles in these sections were histologically abnormal. Disrupted,
nonuniform layers of granulosa cells and numerous pyknotic nuclei,
suggestive of atretic follicles, were present in most sections.
Apoptosis was detected most notably in the granulosa cells of larger,
antral size follicles. By 21–23 weeks of age the phenotype in the ArKO
ovary was more pronounced. Although follicles at all stages were still
observed (Fig. 3C), the typical appearance of closely packed growing
follicles observed in wild-type and heterozygous ovaries (Fig. 2, A–D)
was lost, and many of the follicles present were atretic. Preantral
follicles were randomly scattered throughout the ovary, and many of the
antral follicles that were present were cystic and hemorrhagic (Fig. 3, C and D). The ovaries of 1-yr-old ArKO mice differed vastly in
appearance from the ovaries of age-matched controls. There were no
identifiable secondary or antral follicles in these ovaries (Fig. 4, E and F), although structures that may
represent remnants of large follicles were present (Fig. 3E), many of
which were cystic and hemorrhagic (Figs. 3, E and F, and 4A). In some
cases the cysts constituted the majority of the ovarian space (Fig. 4A). Studies are underway to characterize these degenerative follicular
structures. The primary follicles present were abnormal, consisting of
oddly shaped granulosa cells, many of which were pyknotic. Granulosa
cell apoptosis, as determined by TUNEL assay, was widespread throughout
the ovary (Fig. 4, B and C).
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), the interstitial
regions of the ArKO mouse ovary were disorganized in appearance, with
less densely packed interstitial cells (Fig. 4, G and H). There was
also an increased deposition of collagen with age [scattered collagen
deposits in 10- to 12-week-old animals (Figs. 3, A and B, and 4H)
compared with substantial collagen deposition in 1-yr-old mice (Figs. 3, E and F, and 4 H]. As shown in Fig. 4, H and I, both heterozygote
and young ArKO mice had open oval structures in the interstitial
regions.
Cells hypothesized to be macrophages in the ArKO ovary (Fig. 4D)
were identified using a nonspecific esterase stain (Fig. 4E). In ArKO
ovaries, the numbers of esterase-positive cells increased with age. The
macrophages were generally localized to sites of hemorrhagic cysts or
areas of advanced degeneration within the interstitial region (Figs. 3F and 4D).
Numbers of follicles
As expected, primary follicle numbers were always in excess
of secondary and antral follicle numbers regardless of age or genotype
(Fig. 5). There were no significant
effects of age on the number of follicles in each class in the
wild-type group. ArKO ovaries contained significantly more primary
follicles than wild-type ovaries at 21–23 weeks and more than
heterozygous ovaries at 10–12 and 21–23 weeks (Fig. 5). At 10–12
weeks there were significantly fewer secondary follicles in the ArKO
ovaries, and by 1 yr of age, no identifiable secondary or antral
follicles were present in the ovaries. Ovaries of heterozygotes
contained fewer primary follicles relative to wild-type and ArKO
ovaries at all ages studied, although significance was only obtained at
21–23 weeks (Fig. 5). The secondary and antral follicle numbers of
heterozygotes were consistent with those of the other genotypes.
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). The
nuclear diameters of oocytes within primary follicles of
heterozygous ovaries at 21–23 weeks were greater than those in
the corresponding wild-type follicles. Also at 21–23 weeks, nuclear
diameters of oocytes within secondary and antral follicles of
heterozygotes were greater than those in the corresponding ArKO
mice.
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). The mean diameter of primary
follicles in ovaries of 10- to 12-week-old ArKO mice was significantly
reduced relative to that of wild-type primary follicles at this age.
Primary follicles of 21- to 23-week-old heterozygous ovaries had a
diameter significantly larger than those of the comparable wild-type
and ArKO follicles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the ovaries of ArKO mice lack the capacity to produce
E2, and the animals exhibit a phenotype
consistent with hypoestrogenicity (Ref. 9 and this study), there are
two other sources of estrogenic activity that could impact on the
model. The ovaries of maternal heterozygotes are a source of estrogen,
which could cross the placenta during pregnancy and influence the
development of the fetal ArKO ovary. It is possible that the effects of
maternally derived E2 on the fetal ovary in
utero could still be operative on follicles in the immediate
postnatal period. The extent to which these effects of
E2 are important for normal postnatal
folliculogenesis is unknown. However, by 10–12 weeks of age,
E2 from this source is unlikely to play a role in
folliculogenesis, given the rapid half-life of E2
and the absence of -fetoprotein, an E2 carrier
in rodents (21). We conclude that even if maternal estrogen plays a
role in stimulating ovarian differentiation and primordial follicle
organization, follicular development from the point of primordial
follicle activation up to the antral follicle stage can occur in the
absence of estrogen. A second source of estrogenic activity arises from
the phytoestrogens present in mouse chow (10% soy). Preliminary data
(ovarian and uterine weights) from ArKO mice raised and maintained on a
soy-free diet indicate that these mice exhibit the same, but a more
exacerbated, hypoestrogenic phenotype relative to those ArKO mice
raised and maintained on the soy-containing diet (22). The data suggest
that the onset of the ovarian phenotype was accelerated in ArKO mice
fed soy-free chow.
The data from this study support the conclusion that E2 is required directly or indirectly for normal growth of committed follicles in the mouse. It is not clear as yet whether estrogen is important in determining the size of the primordial follicle pool. Studies in the ArKO mouse are currently in progress to establish the impact of estrogen, if any, at this step in folliculogenesis. The lack of CL and the presence of many large unhealthy antral follicles at 10–12 weeks of age in the ArKO ovaries pinpoint a role for E2 in the progression of follicles from the secondary to the tertiary stage at around the time of selection. At this time proliferation of granulosa cells is maximal, and there is up-regulation of expression of genes in granulosa cells, many of which are regulated by E2 and FSH (2). For example, it has been shown recently that cyclin D2, a critical component of the cell cycle machinery in granulosa cells, is influenced independently by both FSH and E2 (3, 23). Furthermore, E2 is known to modulate the up-regulation of expression of LH receptors by FSH (2, 24). The follicular status of ovaries of ArKO mice suggests a role for E2 in vivo in the growth and differentiation of granulosa cells in secondary follicles under the influence of FSH.
The effect of age on the phenotype of the ArKO ovaries was pronounced.
Cystic and hemorrhagic follicles were prevalent in the ovaries of older
animals, and the primary follicles that remained were atretic. If there
is no change in the exit rate from the primordial pool and yet there is
an absence of secondary and antral follicles at 1 yr of age, this
implies that the oocytes of primary follicles die, resulting in the
loss of follicles of this type from the ovary. It was interesting to
note the distinct ovarian phenotype of the heterozygote mouse. The open
oval structures were similar to the open vacuoles present in GnRH
antagonist-treated ER knockout (ERKO) mice (25). We postulate
that these structures, present in heterozgous and ArKO mice, are the
remains of early stage follicles that have undergone atresia. The
reduced numbers of primary follicles in ovaries of heterozygous mice,
which are evident within a different gonadotropin milieu, add a new
perspective to studies investigating folliculogenesis. The interstitial
areas of the ArKO ovaries were characterized by an apparent decrease in
the density of stromal cells with a more spreadout/disorganized
appearance. This may be due to the collagen deposits in these mice. By
1 yr of age, the interstitial regions of the ArKO ovaries contained
substantial collagen deposition, characteristic of tissues that are
fibrotic and are undergoing degeneration and tissue remodeling. There
was also a massive infiltration of macrophages to the interstitial
region and to sites of follicle degeneration. It is not clear whether
the phenotype observed is due to the lack of estrogen, to the elevated
levels of gonadotropin and testosterone (9), or to both.
The ovarian phenotype in estrogen-deprived ArKO mice has some similarities to other mouse models. Risma et al. (26) provided direct evidence of the effects of chronic exposure to LH on the ovary. They generated transgenic mice overexpressing a LHß transgene in the pituitary; thus, these mice exhibited elevated levels of LH while maintaining normal FSH levels. The ovaries of these transgenic mice contained blood-filled cysts, misshapen granulosa cells, and luteinized cells. The ovaries of these transgenic mice contained 45% fewer primordial follicles at 5 weeks of age, and by 3 months contained decreased numbers of primordial and primary follicles (18), suggesting an increase in primordial follicle cell death. The anovulation observed in these mice was reversed via the administration of a LH-like surge (hCG bolus), suggesting that the anovulation is the result of elevated basal LH levels and the absence of a LH surge (27). Testosterone levels in these mice were 3–5 times higher than those in wild-type animals and may play a significant role in the phenotype observed.
It should be noted that the ovarian morphology of ArKO mice does not resemble that of polycystic ovarian syndrome in humans principally because the thecal layer is not hypertrophied, and there is no pearl drop appearance of follicles. It appears, however, to resemble the ovarian morphology of the ERKO mice, which also had enlarged and hemorrhagic cystic follicles (7). They exhibit elevated LH levels and a termination of follicle development at the antral stage (7). This is consistent with the ArKO model; however, the ovarian phenotype of ERKO mice is apparent at the onset of puberty (20–22 days). No data are available yet for ArKO mice at this age. Additionally, ERKO mice display increased estrogen levels, whereas serum FSH levels are reportedly unaltered (28). The role of estrogen in the pathology observed is complicated by the presence of a second ER, ERß. The generation of an ERß knockout via gene disruption (28, 29) provides evidence for differing roles for the two receptors. Although serum levels of LH and FSH were normal, these mice appeared to be subfertile, with folliculogenesis arrested in some follicles. The ßERKO mouse is dissimilar to the ArKO mouse, in that it remains fertile despite having fewer CLs, which translates into fewer litters with fewer pups (8).
Treatment of ERKO mice with a GnRH antagonist lowered LH levels, returned the ovarian morphology to that comparable with wild-type morphology, and resulted in the absence of cystic follicles (25), demonstrating that the multiple cysts were the result of chronic hyperstimulation by elevated LH. These data are in agreement with those of the LHß transgene overexpression.
Mice lacking both ER and ERß expressed an ovarian phenotype
distinct from those of the ERKO and ßERKO models (30), but at
2.5–7 months of age exhibited some similarities to the ArKO mouse. In
the double ß knockout female, the ovary contained primordial and
growing follicles, some possessing a large antrum, no CL, and elevated
serum LH levels, but no hemorrhagic cyst formation. The researchers
also reported structures resembling seminiferous tubules in these mice
that to date have not been observed in ovaries of ArKO mice.
FSHß-deficient mice are infertile and possess small ovaries lacking
normal follicles beyond the preantral stage and CL (31). These mice
have normal estrogen levels, but elevated LH levels. Interestingly,
these mice do not possess follicular cysts. This model demonstrates
that FSH is not required for the development of ovarian follicles up to
the antral stage. It also suggests a role for elevated LH levels in the
pathogenesis of the ovarian phenotype observed in the ArKO, as the
phenotype observed at 9–12 weeks in the FSHß knockout mice is
similar to that observed in the ArKO mice at the same age.
In conclusion, the ArKO mouse is proving to be a valuable model for investigating the role of estrogen in the folliculogenic process. Dissecting the importance of changes in serum gonadotropins and testosterone, as opposed to a lack of estrogen, in the manifestation of the ArKO ovarian phenotype will be an essential part of future studies.
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Acknowledgments |
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Footnotes |
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Received November 10, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and estrogen receptor-ß (ERß)
messenger ribonucleic acid in the wildtype and ER-knockout mouse.
Endocrinology 138:4613–4621 and ß mRNA expression: impact of development
and estrogen. Mol Cell Endocrinol 149:153–161[CrossRef][Medline]
-fetoprotein concentration in the
serum and its synthesis in the liver of analbuminemic rats. Cancer Res 42:306–308
knockout mouse. Endocrinology 140:5855–5865
and ß. Science 286:2328–2331This article has been cited by other articles:
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D. Tomic, K. P. Miller, H. A. Kenny, T. K. Woodruff, P. Hoyer, and J. A. Flaws Ovarian Follicle Development Requires Smad3 Mol. Endocrinol., September 1, 2004; 18(9): 2224 - 2240. [Abstract] [Full Text] [PDF]
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G. A. Head, V. R. Obeyesekere, M. E. Jones, E. R. Simpson, and Z. S. Krozowski Aromatase-Deficient (ArKO) Mice Have Reduced Blood Pressure and Baroreflex Sensitivity Endocrinology, September 1, 2004; 145(9): 4286 - 4291. [Abstract] [Full Text] [PDF]
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K. L. Britt, P. G. Stanton, M. Misso, E. R. Simpson, and J. K. Findlay The Effects of Estrogen on the Expression of Genes Underlying the Differentiation of Somatic Cells in the Murine Gonad Endocrinology, August 1, 2004; 145(8): 3950 - 3960. [Abstract] [Full Text] [PDF]
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P. Jeyasuria, Y. Ikeda, S. P. Jamin, L. Zhao, D. G. de Rooij, A. P. N. Themmen, R. R. Behringer, and K. L. Parker Cell-Specific Knockout of Steroidogenic Factor 1 Reveals Its Essential Roles in Gonadal Function Mol. Endocrinol., July 1, 2004; 18(7): 1610 - 1619. [Abstract] [Full Text] [PDF]
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M Myers, K L Britt, N G M Wreford, F J P Ebling, and J B Kerr Methods for quantifying follicular numbers within the mouse ovary Reproduction, May 1, 2004; 127(5): 569 - 580. [Abstract] [Full Text] [PDF]
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K. Huynh, G. Jones, G. Thouas, K. L. Britt, E. R. Simpson, and M. E.E. Jones Estrogen Is Not Directly Required for Oocyte Developmental Competence Biol Reprod, May 1, 2004; 70(5): 1263 - 1269. [Abstract] [Full Text] [PDF]
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K. Toda, Y. Okada, M. Zubair, K.-i. Morohashi, T. Saibara, and T. Okada Aromatase-Knockout Mouse Carrying an Estrogen-Inducible Enhanced Green Fluorescent Protein Gene Facilitates Detection of Estrogen Actions in Vivo Endocrinology, April 1, 2004; 145(4): 1880 - 1888. [Abstract] [Full Text] [PDF]
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 A. Belgorosky, C. Pepe, R. Marino, G. Guercio, N. Saraco, E. Vaiani, and M. A. Rivarola Hypothalamic-Pituitary-Ovarian Axis during Infancy, Early and Late Prepuberty in an Aromatase-Deficient Girl Who Is a Compound Heterocygote for Two New Point Mutations of the CYP19 Gene J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5127 - 5131. [Abstract] [Full Text] [PDF]
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K. H. Burns, J. E. Agno, P. Sicinski, and M. M. Matzuk Cyclin D2 and p27 Are Tissue-Specific Regulators of Tumorigenesis in Inhibin {alpha} Knockout Mice Mol. Endocrinol., October 1, 2003; 17(10): 2053 - 2069. [Abstract] [Full Text] [PDF]
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J. F. Couse, M. M. Yates, V. R. Walker, and K. S. Korach Characterization of the Hypothalamic-Pituitary-Gonadal Axis in Estrogen Receptor (ER) Null Mice Reveals Hypergonadism and Endocrine Sex Reversal in Females Lacking ER{alpha} But Not ER{beta} Mol. Endocrinol., June 1, 2003; 17(6): 1039 - 1053. [Abstract] [Full Text] [PDF]
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 K. Toda, T. Okada, C. Miyaura, and T. Saibara Fenofibrate, a ligand for PPAR{alpha}, inhibits aromatase cytochrome P450 expression in the ovary of mouse J. Lipid Res., February 1, 2003; 44(2): 265 - 270. [Abstract] [Full Text] [PDF]
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 K. L. BRITT, J. KERR, L. O'DONNELL, M. E. E. JONES, A. E. DRUMMOND, S. R. DAVIS, E. R. SIMPSON, and J. K. FINDLAY Estrogen regulates development of the somatic cell phenotype in the eutherian ovary FASEB J, September 1, 2002; 16(11): 1389 - 1397. [Abstract] [Full Text] [PDF]
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K. H. Burns and M. M. Matzuk Minireview: Genetic Models for the Study of Gonadotropin Actions Endocrinology, August 1, 2002; 143(8): 2823 - 2835. [Abstract] [Full Text] [PDF]
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N. Danilovich and M. R. Sairam Haploinsufficiency of the Follicle-Stimulating Hormone Receptor Accelerates Oocyte Loss Inducing Early Reproductive Senescence and Biological Aging in Mice Biol Reprod, August 1, 2002; 67(2): 361 - 369. [Abstract] [Full Text] [PDF]
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N. Danilovich, D. Javeshghani, W. Xing, and M. R. Sairam Endocrine Alterations and Signaling Changes Associated with Declining Ovarian Function and Advanced Biological Aging in Follicle-Stimulating Hormone Receptor Haploinsufficient Mice Biol Reprod, August 1, 2002; 67(2): 370 - 378. [Abstract] [Full Text] [PDF]
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Z. Fang, S. Yang, B. Gurates, M. Tamura, E. Simpson, D. Evans, and S. E. Bulun Genetic or Enzymatic Disruption of Aromatase Inhibits the Growth of Ectopic Uterine Tissue J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3460 - 3466. [Abstract] [Full Text] [PDF]
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G. Cheng, Z. Weihua, S. Makinen, S. Makela, S. Saji, M. Warner, J.-A. Gustafsson, and O. Hovatta A Role for the Androgen Receptor in Follicular Atresia of Estrogen Receptor Beta Knockout Mouse Ovary Biol Reprod, January 1, 2002; 66(1): 77 - 84. [Abstract] [Full Text]
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L. D. Quirke, J. L. Juengel, D. J. Tisdall, S. Lun, D. A. Heath, and K. P. McNatty Ontogeny of Steroidogenesis in the Fetal Sheep Gonad Biol Reprod, July 1, 2001; 65(1): 216 - 228. [Abstract] [Full Text] [PDF]
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S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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) localized in
the Masson’s Trichrome-stained 1-yr-old ArKO ovary (magnification,
x100). E, Nonspecific esterase staining of 1-yr-old ArKO ovaries
(magnification, x100), showing esterase-positive cells (e+), as
depicted by brown-red granular staining. F, Interstitial
region of the 10- to 12-week-old wild-type ovary, with a CL and
interstitial cells (magnification, x200). G, Interstitial region of
10- to 12-week-old ArKO ovary. Open oval structures (OO) and collagen
deposits (CD) were observed (magnification, x100). H, Interstitial
region of 1-yr-old ArKO (magnification, x200). Note the
blue appearance of the ovary, representing an increased
collagen deposition (C). I, Open oval structures (OO) found in 10- to
12-week-old heterozygous ovary (magnification, x200).
