BIOLOGY OF REPRODUCTION 76, 1045–1053 (2007)
Published online before print 28 February 2007.
DOI 10.1095/biolreprod.106.059360
Role of Central Nervous System and Ovarian Insulin Receptor Substrate 2 Signaling in
Female Reproductive Function in the Mouse1
Irina Neganova,3 Hind Al-Qassab,3 Helen Heffron,3 Colin Selman,3 Agharul I. Choudhury,3
Steven J. Lingard,3 Ivan Diakonov,3 Michael Patterson,4 Mohammad Ghatei,4 Stephen R. Bloom,4
Stephen Franks,5 Ilpo Huhtaniemi,5 Kate Hardy,5 and Dominic J. Withers2,3
Centre for Diabetes and Endocrinology,3 Rayne Institute, University College London, London WC1E 6JJ, United Kingdom
Department of Metabolic Medicine,4 and Institute of Reproductive and Developmental Biology,5 Imperial College
London, London W12 0NN, United Kingdom
ABSTRACT
Insulin receptor signaling regulates female reproductive
function acting in the central nervous system and ovary. Female
mice that globally lack insulin receptor substrate (IRS) 2, which
is a key mediator of insulin receptor action, are infertile with
defects in hypothalamic and ovarian functions. To unravel the
tissue-specific roles of IRS2, we examined reproductive function
in female mice that lack Irs2 only in the neurons. Surprisingly,
these animals had minimal defects in pituitary and ovarian
hormone levels, ovarian anatomy and function, and breeding
performance, which indicates that the central nervous system
IRS2 is not an obligatory signaling component for the regulation
of reproductive function. Therefore, we undertook a detailed
analysis of ovarian function in a novel Irs2 global null mouse
line. Comparative morphometric analysis showed reduced
follicle size, increased numbers of atretic follicles, as well as
impaired oocyte growth and antral cavity development in Irs2
null ovaries. Granulosa cell proliferation was also defective in
the Irs2 null ovaries. Furthermore, the insulin- and eCGstimulated phosphoinositide-3-OH kinase signaling events,
which included phosphorylation of Akt/protein kinase B and
glycogen synthase kinase 3-beta, were impaired, whereas
mitogen-activated protein kinase signaling was preserved in
Irs2 null ovaries. These abnormalities were associated with
reduced expression of cyclin D2 and increased CDKN1B levels,
which indicates dysregulation of key components of the cell
cycle apparatus implicated in ovarian function. Our data suggest
that ovarian rather than central nervous system IRS2 signaling is
important in the regulation of female reproductive function.
insulin, insulin-like growth factor receptor, kinases, oocyte
development, ovary
INTRODUCTION
Insulin receptor (INSR) signaling pathways regulate peripheral energy homeostasis by acting in skeletal muscle, adipose
tissue, and the liver to control carbohydrate, lipid, and protein
metabolism. It is clear that these signals also act in other
1
Supported by an MRC Co-operative Component Grant and a Wellcome Trust Functional Genomics Award.
2
Correspondence: Dominic J. Withers, Centre for Diabetes and
Endocrinology, Rayne Institute, 5 University Street, University College
London, London WC1E 6JJ, United Kingdom. FAX: 4420 767 96583;
e-mail: [email protected]
Received: 7 December 2006.
First decision: 23 December 2006.
Accepted: 19 February 2007.
Ó 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
tissues, for example, in the regulation of pancreatic b-cell and
hypothalamic functions [1]. Insulin receptor signaling has also
been implicated in the regulation of female reproductive
function through its actions in both the central nervous system
(CNS) and ovaries. Female mice that lack insulin receptors in
their neurons show impaired fertility with fewer antral follicles
and corpora lutea due to reduced release of LH [2]. Insulin also
stimulates GnRH secretion in vivo, and intracerebroventricular
administration of insulin restores reproductive behavior in
diabetic rats [3, 4]. In vitro studies using immortalized GnRH
neuronal cell lines have suggested that insulin regulates GnRH
expression through the mitogen-activated protein kinase
pathway [5]. Therefore, CNS insulin signaling plays a role in
regulating female reproductive function in rodents. Insulin
signaling has also been shown to have complex roles in ovarian
function, including the regulation of ovarian steroidogenesis,
follicular development, and granulosa cell proliferation [6–8].
The strong association of insulin resistance with ovarian
dysfunction in polycystic ovarian syndrome also suggests a
role for insulin signaling in ovarian function [9].
Insulin receptor substrate (IRS) proteins mediate the effects
of the insulin receptor on cellular and whole body physiology,
including reproduction [10]. Mice that lack Irs1 display
profound growth retardation and insulin resistance but have
mildly defective reproductive function [11, 12]. Irs3 and Irs4
null mice have minimal metabolic, endocrine, and growth
phenotypes [10]. In contrast, mice that lack Irs2 develop
diabetes due to a combination of insulin resistance and
pancreatic b cell dysfunction [11]. Female Irs2 null mice are
also infertile due to reduced pituitary LH levels and
gonadotroph cell numbers, and show reduced gonadotropinstimulated ovulation and markedly reduced numbers of ovarian
follicles and corpora lutea [12]. While CNS IRS2 signaling
plays complex roles in energy homeostasis [1], the contribution
of CNS IRS2 to female reproductive function has not been
established. Ovarian cellular defects and downstream signaling
abnormalities in Irs2 null mice have not been characterized.
Therefore, we used conditional gene targeting in mice to delete
Irs2 in the CNS and examined their reproductive functions. In
addition, we generated a novel Irs2 null mouse line and
assessed its ovarian morphology, signaling, and cell cycle
events.
MATERIALS AND METHODS
Ethics
All of the in vivo studies were performed in accordance to the United
Kingdom Home Office Animal Procedures Act (1986) and University College
London Animal Ethical Committee Guidelines.
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NEGANOVA ET AL.
Experimental Animals
Mice with a floxed allele of Irs2 (Irs2tm1With) and mice that lack Irs2 in the
CNS, Irs2tm1With /Irs2tm1With Tg (Nes-cre)1Kln/0 (hereinafter designated as
NesCreIrs2KO mice) have been described previously [1]. Mice with a germline deletion of Irs2 were also generated using embryonic stem cells that had
undergone complete deletion of the Irs2 locus following Cre recombinase
treatment during the same targeting strategy [1]. The mice were back-crossed
five times onto a C57Bl/6 background prior to analysis. Genotyping was
performed as previously described [1]. The mice were maintained on a
12L:12D cycle with free access to water and standard mouse chow (4% fat,
RM1; Special Diet Services, Witham, Essex, UK) and housed in specificpathogen-free barrier facilities.
In Vivo Analyses of Reproductive Function
Estrus cycle characteristics were determined by analysis of vaginal smears
for cell type and morphology with Giemsa staining each day for three complete
cycles. Fertility was assessed by monitoring daily for 3 mo the numbers of
pregnancies, litters, and pups in continuous mating studies with confirmed male
breeders and 7–9-week-old females.
Hormone Stimulation, Sample Collection, and Hormone
and Peptide Measurements
For the superovulation studies, 8–10-wk-old mice received 10 IU eCG
(Folligon; Intervet) by i.p. injection, followed 46 h later by 8 IU hCG (Intervet).
Oocytes were flushed from the oviduct 14–16 h after the second injection. For
bromodeoxyuridine (BrdU) incorporation studies, the mice received an i.p.
injection of 100 mg/kg BrdU (Roche) 2 h before tissue harvesting. Blood
samples were collected by tail vein bleeding or by cardiac puncture of
terminally anesthetized mice at specific stages of the estrus cycle, and the serum
was either stored at 808C prior to analysis or allowed to coagulate at 48C
overnight, centrifuged, and frozen at 208C. Pituitaries were harvested,
homogenized in 1 M urea in PBS, and stored at 808C prior to analysis. Serum
estrogen and progesterone were measured using immunofluorometric assays
(Delfia; Perkin Elmer, Turku, Finland). RIAs for pituitary LH, FSH, and
prolactin were performed as previously described [13, 14].
Histomorphometric Analyses and Immunohistochemistry
Ovaries were fixed in 10% buffered formalin, embedded in paraffin, and cut
into 5-lm sections. For histomorphometric analysis, sections were stained with
hematoxylin-eosin. Follicles, which were classified according to diameter and
number of granulosa cell layers, were counted in every tenth section, starting
from the fifth section, through the entire ovary. Only those follicles with a
clearly visible oocyte nucleus were counted, to avoid double counting. Atretic
follicles were defined according to the following morphological criteria: follicle
and oocyte shapes, localization of the oocyte in the follicle, presence of a
degenerated oocyte, altered zona pellucida, the presence of granulosa cells
inside the zona pellucida, the presence of more than three pyknotic nuclei in the
granulosa cells, and macrophage invasion. Follicle growth, follicle and oocyte
size (diameter), and total follicular and antral cavity area were measured using
the LUCIA software (Nikon UK, Kingston-upon-Thames, UK). For immunohistochemistry, sections were deparaffinized and antigen retrieval was
performed by microwave boiling of the sections in 10 mM citrate buffer (pH
6.0) twice for 10 min each. Sections were incubated in 20% (v/v) normal goat
serum in PBS with 4% (w/v) BSA for 30 min at room temperature, and then
with the indicated primary antibodies overnight at 48C. Detection of bound
antibodies was performed using the avidin-biotin complex method (Vectastain
ABC Elite kit, Vector Laboratories). Sections were also counterstained with
hematoxylin, dehydrated in a graded ethanol series, cleared in xylene, and
mounted. The Zymed proliferation cell nuclear antigen (PCNA) and BrdU
detection kits were used for PCNA and BrdU staining, respectively.
Western Blotting
Mice (8–10 wk of age) were either superovulated as described above or
treated with insulin as described previously [15]. In brief, mice were anesthetized
and injected i.v. with saline or 5 U of soluble insulin, and the ovaries were
removed and snap frozen 10 min later. Tissue preparation and lysis, Western
blotting, and quantification were performed as previously described [15]. The
blots were stripped and probed for actin when loading controls were required.
Antibodies
Affinity-purified rabbit anti-IRS1 and anti-IRS2 antibodies were from
Upstate Biotechnology (Dundee, UK). The anti-AKT, anti-pAKTSer473, anti-
glycogen synthase kinase (GSK)3B, anti-pGSK3A/BSer9/21, anti-mitogenactivated protein kinase (MAPK) and anti-pMAPKThr202/Tyr204 antibodies were
from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibodies
to cyclin D2 and CDKN1B (p27KIP1) and mouse anti-actin were from Santa
Cruz Biotechnology (Insight Biotechnology, Wembley, UK).
Real-Time PCR
Real-time PCR was performed as previously described using FAM/
TAMRA primers (Applied Biosystems, Foster City, CA) [15]. The following
primer sets were used: Irs1 Mm00439720_s1, Irs2 (MIRS 396412), and Hprt1
Mm01545399_m1. The relative amounts of mRNA were calculated from an
internal standard curve following normalization to the Hprt1 mRNA levels.
Statistical Analysis
All statistical analyses were performed using the GraphPad Prism4 software
(GraphPad Software, San Diego, CA), and paired and unpaired t-tests and twoway ANOVA with Bonferroni post-hoc tests were performed as appropriate. P
, 0.05 was regarded as statistically significant.
RESULTS
Reproductive Phenotype of NesCreIrs2KO Mice
NesCreIrs2KO mice lack Irs2 expression in the CNS and
hypothalamus [1], while their expression of pituitary and
ovarian Irs2 mRNA is equivalent to that in control animals,
i.e., the relative mRNA levels expressed as percentages of the
wild-type (WT) control are: Irs2 in NesCreIrs2KO pituitaries,
98.65 6 12.5%; Irs2 in NesCreIrs2KO ovaries, 107.4 6
15.2%; n ¼ 12, P . 0.05. There were no changes in Irs1
expression in the ovary, hypothalamus, and pituitary of
NesCreIrs2KO mice (data not shown). Therefore NesCreIrs2KO mice represent a good model for determining the
contribution of CNS IRS2 pathways to female reproductive
function. The NesCreIrs2KO mice exhibited an extended estrus
cycle due to a mildly prolonged diestrus phase (Table 1).
Diestrus pituitary prolactin levels were reduced in NesCreIrs2KO mice, whereas the serum estradiol and progesterone and
pituitary FSH and LH levels were similar to those of WT mice
(Table 1). No differences in the timing of ovulation and the
numbers of oocytes recovered were seen when NesCreIrs2KO
mice and WT mice were superovulated (total oocytes
recovered per animal: WT, 23.6 6 2.3 vs. NesCreIrs2KO
21.1 6 2.1, n ¼ 5, P . 0.05). Morphological studies showed
that follicles at all developmental stages and corpora lutea were
present in these animals and in WT mice, which is consistent
with their normal responses to superovulation (Fig. 1, A and B
and data not shown). In continuous mating studies, the average
duration between litters and the size and number of litters over
the study period were similar in NesCreIrs2KO and WT mice
(Table 1). NesCreIrs2KO mice were able to suckle and rear
their progeny and there were no differences in the postpartum
survival rates of these mice compared to WT females (data not
shown). Therefore, somewhat surprisingly, deletion of Irs2 in
the CNS has a minimal effect on reproductive function in mice.
A previous analysis of ovarian function in Irs2 null mice
was limited to the demonstration of reduced numbers of
ovarian follicles and corpora lutea, defective ovarian responses
to superovulation, and reduced circulating gonadal steroid
levels [12]. There are currently limited options for ovarianspecific gene deletion throughout folliculogenesis. Therefore,
we examined in detail the ovarian morphology and ovarian cell
proliferation and we analyzed the signaling events and cell
cycle components downstream of IRS2 in our novel Irs2 null
mouse on a C57Bl/6 background.
After superovulation, reduced numbers of oocytes were
recovered from 8–10-week-old Irs2 null mice (total oocytes
IRS2 REGULATES OVARIAN FUNCTION
1047
TABLE 1. Reproductive characteristics of NesCreIrs2KO mice.
Characteristic
Estrous cycle (n ¼ 9)a
Proestrus (days)
Estrus (days)
Metestrus (days)
Diestrus (days)
Total duration (days)
Hormone measurement (n ¼ 7–13)a
Estradiol (nmol/L)
Progesterone (nmol/L)
LH (ng/ml)
Prolactin (ng/ml)
FSH (ng/ml)
Breeding performance (n ¼ 6)a
Litters
Pups
Pups per litter
Litter per female
Durations between litters (days)
a
b
c
WTb
1.2
1.3
1.2
1.2
4.9
6
6
6
6
6
0.1
0.2
0.1
0.1
0.1
0.016
4.198
300.6
4048
46.07
6
6
6
6
6
0.002
0.7
37.3
840.7
2.8
23
172
7.5 6 0.7
3.8 6 0.2
26.1 6 1.9
NesCreIrs2KOb,c
1.5
1.3
1.4
1.9
6.1
6
6
6
6
6
0.2
0.2
0.2
0.2*
0.2*
0.017
3.43
218.5
979.5
36.63
6
6
6
6
6
0.04
1.0
34.3
126.9**
4.2
19
131
7.2 6 0.7
3.2 6 0.3
28.7 6 2.7
n ¼ Number of mice per genotype.
All results are presented as means 6 SEM.
Values are significantly different; *P , 0.05, **P , 0.01.
recovered per animal: WT, 23.6 6 2.3 vs. Irs2 null 8.3 6 0.8,
n ¼ 5, P , 0.01). Irs2 null ovaries showed increased numbers
of atretic follicles, reduced numbers of large antral follicles,
and an absence of preovulatory follicles (Fig. 1, C and D).
Additional characteristic features of Irs2 null ovaries were the
presence of oocytes trapped in corpora lutea (Fig. 1E) and the
presence of occasional follicles with double oocytes (Fig. 1F).
We explored further the mechanisms underlying these defects.
IRS2 and IRS1 Protein Expression by Ovarian Cells
Strong specific IRS2 staining was seen in oocytes and
granulosa cell cytoplasm of the early primordial follicles in the
WT mice but not in the Irs2 null animals (Fig. 2, A–D). Lessextensive IRS2 staining was detected in the thecal and stromal
cells at all stages of follicular development in the WT mice
(Fig. 2, A–C). IRS1 was expressed in a range of ovarian cell
types in the WT mice but not in the Irs1 null mice (Fig. 2, E–
H). Staining was more intense in the granulosa cells than in
thecal cells and increased with follicular growth. In the Irs2
null mice, there was an apparent reduction in the expression of
IRS1 protein in the ovary (Fig. 2, I–K). This was confirmed by
the demonstration of reduced Irs1 mRNA expression in Irs2
null ovaries (relative Irs1 mRNA levels expressed as
percentage of the WT control: 73 6 2%, n ¼ 3, P , 0.001).
These findings suggest that IRS2 plays complex roles in
ovarian cellular function. Therefore, we characterized in detail
the follicular morphologies of the WT and Irs2 null mice.
Analysis of Follicular Development in Irs2 Null Ovaries
Morphometric analysis confirmed a marked reduction in the
number of follicles of all sizes in Irs2 null ovaries and that 75%
of the large antral follicles were atretic in Irs2 null mice (Fig. 3,
A and B). When there were fewer than three follicular
granulosa cell layers, no differences were observed between the
development of these follicles in Irs2 null and WT mice (Fig.
3C). However, the follicles at later stages were reduced in size
(Fig. 3C), which suggests that Irs2 null ovaries are defective
for granulosa cell proliferation within growing follicles.
Complex interactions occur between oocytes and other
follicular components, with oocytes promoting follicular
FIG. 1. Ovarian anatomies of WT, NesCreIrs2KO, and Irs2 null mice. A–
C) Low magnification photomicrographs of typical WT (A), NesCreIrs2KO
(B), and Irs2 null (C) ovarian morphologies. In C, the arrows indicate
atretic follicles. D–F) High magnification of photomicrographs of Irs2 null
ovaries demonstrating atretic follicles (D), oocytes trapped in corpora
lutea (E; arrow), and a follicle that contains a double oocyte (F). Bar ¼ 0.5
mm (A-C) or 100 lm (D–F). The brown staining is peroxidase staining
from the immunohistochemistry shown in detail in subsequent Figures.
growth and directing granulosa cell differentiation [16–19].
In Irs2 null mice, the early oocytes grew as quickly as those in
the WT mice but growth was impaired at later stages (Fig. 3D).
Antral cavity development and expansion were also markedly
attenuated in Irs2 null ovaries (Fig. 3E). After superovulation
in the Irs2 null mice there was a minimal increase in antral
cavity size (data not shown). These morphological parameters
demonstrate that in Irs2 null follicles, three events that are
important for normal follicular development from the growing
pool to the preovulatory stage are markedly disturbed.
Granulosa Cell Proliferation in Irs2 Null Follicles
We used PCNA expression and BrdU incorporation to
assess further cell proliferation. In the WT ovaries, nuclear
PCNA staining was seen in cuboidal granulosa cells in the
primary follicles and in most of the granulosa cells of follicles
at later stages (Fig. 4, A–D). Thecal cell staining was seen as
the follicles developed granulosa cell layers. Oocytes were
PCNA positive in the primary follicles onwards (Fig. 4A). In
antral follicles, PCNA was expressed in all the granulosa cell
layers, with the highest levels seen in those around the oocyte
(Fig. 4C). Superovulation induced preovulatory follicles with
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NEGANOVA ET AL.
FIG. 2. Localization of IRS2 and IRS1 in
ovarian follicles. A–C) IRS2 immunostaining
of WT ovaries in primordial and primary
follicles (A), secondary follicles (B), and
granulosa cells (C) of early antral follicles.
D) IRS2 staining is absent from the Irs2 null
ovaries. E–G) IRS1 immunostaining of WT
ovaries in primordial and primary follicles
(E), secondary follicle and thecal cells (F),
and in a large preantral follicle (G). H) Lack
of IRS1 staining in an Irs1 null ovary. I–K)
IRS1 staining of Irs2 null ovary demonstrates
decreased expression of IRS1 in the primary
(I), secondary (J), and large preantral
follicles (K). L) There is no staining when the
IRS1 antibody is omitted in Irs2 null ovary
staining. Bar ¼ 100 lm.
strong cumulus PCNA staining (Fig. 4D). In contrast, Irs2 null
ovaries displayed a marked reduction in granulosa cell PCNA
staining in all follicular classes (Fig. 4, E–H). After
superovulation, there was little PCNA staining in the Irs2 null
ovaries, which suggests that the vast majority of granulosa cells
in Irs2 null ovaries do not proceed to a rapid phase of
proliferation (Fig. 4H).
To assess S phase progression, we analyzed BrdU
incorporation in the ovaries. In small follicles with two or
fewer granulosa cell layers, there were no differences in the
numbers of BrdU positive cells in the WT and Irs2 null
ovaries. In the WT mice, increased follicle size was associated
with a marked increase in BrdU labeling (Fig. 4I). In the large
antral follicles, there was a general reduction in BrdU staining
but the cumulus cells showed a high frequency of positive
staining (Fig. 4J). BrdU labeling was reduced in Irs2 null
follicles of all sizes (Fig. 4, K and L). Superovulation
increased BrdU labeling in Irs2 null follicles but to a
significantly lesser extent to that seen in WT ovaries
(percentage of BrdU-positive cells in secondary follicles
(WT vs. Irs2 null): without stimulation, 13.8 6 2.3 vs. 3.6 6
0.9, n ¼ 6, P , 0.05: after superovulation, 19.3 6 3.4 vs. 1.7
6 1.2, n ¼ 6, P , 0.05; and percentage of BrdU-positive cells
in the large antral follicles: without stimulation, 26.9 6 2.1
vs. 9.6 6 0.3, n ¼ 6, P , 0.05; after superovulation, 33.1 6
3.3 vs. 17.8 6 2.8, n ¼ 6, P , 0.05).
Defective PI3 Kinase Signaling Pathways and Abnormal
Expression of Cell Cycle Regulators Cyclin D2 and
CDKN1B in Irs2 Null Ovaries
IRS2 recruits the PI3 kinase/AKT and MAP kinase
pathways, which together mediate the effects of insulin and
IGF1 [10]. These downstream signaling events were not
examined in the Irs2 null mice described previously [12].
Insulin induced a 5-fold increase in Akt phosphorylation in WT
ovaries, which was indicative of PI3 kinase pathway activation,
although this was largely abrogated in Irs2 null ovaries (Fig. 5,
A and B). Treatment with hCG stimulated AKT phosphorylation in the ovaries of mice of either genotype but both the
basal and peak-stimulated AKT phosphorylation were attenuated in Irs2 null ovaries, despite similar levels of AKT
expression (Fig. 5, C and D). AKT phosphorylates and
inactivates glycogen synthase kinase-3 b (GSK3B) [20, 21],
which in turn phosphorylates cyclin D, thereby targeting it for
degradation [22]. Insulin increased GSK3B phosphorylation in
the WT ovaries but not in the Irs2 null ovaries (Fig. 5, E and
F). In contrast, insulin-stimulated MAP kinase activation was
equivalent in the ovaries of both genotypes and hCGstimulated MAP kinase activation was also largely preserved
(Fig. 5, G and H and data not shown).
We also examined the expression levels of cyclin D2, which
is a critical component of the cell cycle apparatus that regulates
G1/S phase progression, and the cell cycle inhibitor CDKN1B.
IRS2 REGULATES OVARIAN FUNCTION
1049
The basal expression of cyclin D2 was reduced and that of
CDKN1B was elevated in Irs2 null ovaries compared to WT
ovaries, which implies an imbalance between these cell cycle
regulators (Fig. 5, I–L). Superovulation induction with eCG
followed by hCG injection caused a significant reduction in
cyclin D2 expression 24 h after hCG treatment (Fig. 5, I and J).
This effect was significantly impaired in Irs2 null ovaries. The
same treatment induced a significant increase in CDKN1B
expression in the WT ovaries but no significant alteration in
Irs2 null ovaries, which also demonstrated a higher basal level
of CDKN1B (Fig. 5, K and L).
Primary follicles from mice of both genotypes showed no
differences in cyclin D2 immunostaining, with a strong pattern
of staining seen in most granulosa cells (Fig. 6, A and F). In the
subsequent stages of follicular growth, both the numbers of
granulosa cells that expressed cyclin D2 and the intensity of
staining were reduced in Irs2 null compared to WT ovaries
(Fig. 6, B–E and G–J). In WT ovaries, 46 h after the
administration of eCG alone, most of the stained cells were
granulosa cells surrounding the oocyte (Fig. 6C). In large
preovulatory follicles (with or without prior superovulation
induction) most of the stained granulosa cell were cumulus
cells, whereas the cells in the mural granulosa were mostly
unstained (Fig. 6, D and E). These results are consistent with
the patterns of PCNA staining and BrdU incorporation
described above. In contrast, in the Irs2 null ovaries in which
large antral follicles were found, few cells expressed cyclin D2
(Fig. 6, I and J), which suggests that impaired proliferation of
cumulus cells contributes to defective ovulation in these mice.
High levels of CDKN1B expression were found in the
corpora lutea of the WT mice, indicating that these cells were
no longer dividing (Fig. 6K). In contrast, most of the granulosa
cells from growing small and antral follicles did not express
CDKN1B (Fig. 6, K–O). In the WT animals, eCG stimulation
did not induce the expression of CDKN1B in granulosa and
cumulus cells (Fig. 6M). However, in Irs2 null ovaries, some
positive staining was observed from the stage of small antral
follicles (Fig. 6Q). Moreover, 46 h after eCG stimulation,
CDKN1B staining was observed in cumulus cells and in mural
and thecal cells in the large antral follicles (Fig. 6R). After
superovulation of the WT mice, no CDKN1B staining was seen
in the cumulus cells and the majority of the mural granulosa
cells (Fig. 6, N and O). This is consistent with the high level of
cyclin D2 expression seen 8.5 h after hCG treatment in the
cumulus cells in the preovulatory follicles of the WT mice (Fig.
6, D and E). In contrast, in Irs2 null ovaries, the granulosa cells
showed positive staining for CDKN1B 46 h after eCG injection
(Fig. 6R). After administration of hCG, positive staining for
CDKN1B was seen in the cumulus cells, suggesting that
proliferation was arrested in these cells (Fig. 6, S and T).
DISCUSSION
Insulin signaling pathways in the CNS have been implicated
in the regulation of female reproductive function [2, 10].
Insulin receptor substrate proteins 1 and 2 are the major
downstream effector molecules recruited by the activated
insulin receptor [10]. Female Irs2 null mice are infertile, with
3
FIG. 3. Comparative analysis of the ovarian morphologies of WT and Irs2
null mice. A) Distribution of follicles according to diameter (lm) and total
number of atretic follicles in WT and Irs2 null ovaries. B) Proportion of
normal and atretic follicles of different diameters (lm) in WT and Irs2 null
follicles. C) Effect of Irs2 deletion on ovarian follicle growth. Each bar
represents the mean 6 SEM (n ¼ 6 ovaries per genotype); *, P , 0.05. D)
Effect of Irs2 deletion on oocyte growth. E) Effect of Irs2 deletion on antral
cavity development during follicular growth. Values represent means 6 SEM
and represent data from 194 WT and 173 Irs2 null follicles. *, P , 0.05.
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NEGANOVA ET AL.
defects in hypothalamic and ovarian functions, which suggest
that IRS2 mediates the effects of the insulin receptor on
reproductive function in a number of tissues [12]. Furthermore,
since Irs2 null mice display defects in leptin action, IRS2 may
integrate the actions of insulin and leptin on energy
homeostasis and fertility [12]. Surprisingly, NesCreIrs2KO
mice, which lack Irs2 in all their neurons, displayed minimal
reproductive abnormalities with almost normal hormone levels,
ovarian functions, and fertility rates. There are several possible
explanations for these findings. IRS1 may compensate in the
CNS of NesCreIrs2KO mice, although we detected no
alterations in the expression of Irs1 in the hypothalami of
these animals. The timing of deletion of Irs2 is different in Irs2
null and NesCreIrs2KO mice, as the nestin transgene is
expressed from E9 onwards, whereas Irs2 expression never
occurs in the global null animal. Therefore subtle developmental differences may explain the phenotype. Taken together,
these results and the demonstration of marked hyperphagia and
obesity in NesCreIrs2KO mice suggest that CNS Irs2
expression is required for the effects of insulin on energy
balance but not on reproductive function. Furthermore, we
have demonstrated that leptin action in NesCreIrs2KO mice is
normal, which suggests that IRS2 is not an obligatory point of
convergence for the actions of insulin and leptin [1].
Irs2 was expressed in a number of ovarian cell types, with
high levels in the follicular granulosa cells, which suggests that
defective insulin and IGF1 actions in these cells may account
largely for the infertility phenotype of Irs2 null mice. Analyses
of follicular development and growth demonstrated a series of
interrelated abnormalities in these animals. While early
follicular development and oocyte development were not
different between the WT and Irs2 null mice, once the follicles
had reached the stage in which growth became gonadotropindependent there was a marked decrease in their numbers. There
was a significant reduction in the number and size of antral
follicles due to reduced antral cavity development and
granulosa cell proliferation and an increase in follicular atresia.
Normally, in primordial follicles, the oocyte is surrounded by a
single layer of nondividing cells that are arrested in G0; these
cells can be induced by appropriate stimuli to undergo
proliferation. Our studies demonstrate that in primordial and
small follicles, cell proliferation is largely preserved in Irs2 null
ovaries. However, the rapid phase of follicular growth that
occurs when granulosa cells acquire enhanced FSH and LH
responsiveness and produce estrogen was abrogated in Irs2 null
ovaries. Terminal differentiation (luteinization), which occurs
when the LH surge causes granulosa cells in preovulatory
follicles to exit the cell cycle, was also abnormal in the Irs2 null
ovaries. Taken together, these findings demonstrate disruption
of the co-ordinated growth pattern within follicles and loss of
3
FIG. 4. Expression of PCNA and BrdU incorporation in WT and Irs2 null
follicles. A–D) WT ovarian PCNA expression in primordial (thick arrow)
and primary follicles (indicated by a short arrow in thecal cell and by a
long arrow in granulosa cells) (A), small transitional follicle granulosa and
thecal cells (B), granulosa and thecal cells in a large antral follicle (C), and
in cumulus cells (D) surrounding an oocyte 8.5 h after superovulation
induction. E–H) PCNA expression in Irs2 null ovaries in a primary follicle
(E), small transitional follicle (F), granulosa cells of a large follicle (G), and
low-level expression in granulosa cells (H) surrounding an oocyte 8.5 h
after hCG treatment. I, J) WT ovarian BrdU incorporation in a transitional
follicle (I) and in cumulus cells of a preovulatory follicle after
superovulation (J). K, L) Irs2 null ovarian BrdU incorporation in a
transitional follicle (K) and after superovulation induction (L). Sections
shown are representative of ovaries from six animals of each genotype and
demonstrate typical patterns of PCNA and BrdU staining. Bar ¼ 100 lm.
IRS2 REGULATES OVARIAN FUNCTION
1051
FIG. 5. Phosphorylation of AKT, GSK3B, and MAP kinase and expression of cyclin D2 and CDKN1B in WT and Irs2 ovaries. A) Western blot of phosphoAKT and total AKT in insulin-stimulated ovarian lysates from WT and Irs2 null mice. B) Densitometric quantification of A. C) Western blot of phospho-AKT
and total AKT in hCG-stimulated ovarian lysates from WT and Irs2 null mice. D) Densitometric quantification of C. E) Western blot of phospho-GSK3B and
total GSK3B in insulin-stimulated ovarian lysates from WT and Irs2 null mice. F) Densitometric quantification of E. G) Western blot of phospho-MAP
kinase and total MAP kinase in insulin-stimulated ovarian lysates from WT and Irs2 null mice. H) Densitometric quantification of G. I and K) Western blots
for cyclin D2 (I) and CDKN1B (K) in ovarian lysates from basal and hCG-stimulated WT and Irs2 null mice. Actin loading controls are also presented. J and
L) Densitometric quantification of I and K, respectively. Representative blots are shown and quantification represents the mean 6 SEM of results obtained
in three independent experiments. *, P , 0.05.
the bidirectional communication between oocytes and granulosa cells during follicle development.
The defects seen in our novel Irs2 null mouse model were not
as severe as those described in the original knockout mouse.
Differences in genetic background are likely to account for this
finding, with the current model using mice on a C57Bl/6
background, while the earlier studies were performed in a mixed
129Sv/C57Bl/6 strain. Consistent with this observation are the
findings that the metabolic phenotype of Irs2 null mice is
strongly influenced by genetic background, and that the fecundity
of C57Bl/6 mice is greater than that of 129Sv mice [23].
IRS2 lies downstream of both INSR and IGF1R and is able to
recruit the MAP kinase and PI3 kinase signaling cascades,
which have been implicated in ovarian function [6–8, 10].
However, we found that MAP kinase expression and phosphorylation in response to insulin or hCG were largely
preserved in Irs2 null ovaries. Other signaling components,
such as IRS1 and SHC, may be involved in the recruitment and
activation of the MAP kinase in Irs2 null ovaries, although
normal MAP kinase activity in response to these hormones is
insufficient to preserve follicular function in these mice. In
contrast, we found significant signaling defects in the PI3 kinase
cascade in Irs2 null ovaries, with both insulin and hCG
stimulation of AKT being impaired. Previous studies have
implicated PI3 kinase/AKT signaling in the maintenance of the
preovulatory follicle granulosa layers and have demonstrated
that FSH induces a biphasic increase in AKT phosphorylation in
rat granulosa cells [24]. We have shown that the metabolic
effects of FSH in the mouse cumulus-oocyte complexes are
mediated by the PI3 kinase pathway [25]. However, insulin
appears to stimulate a larger increase in AKT serine phosphorylation than LH/hCG in the rat ovary in vivo [26]; our data from
mouse ovaries are consistent with this observation.
A number of substrates of activated AKT that regulate cell
survival and proliferation have been identified, including
components of the apoptotic machinery, such as caspase 9
and BAD, which may be involved in IGF1-mediated protection
of granulosa cell apoptosis [27, 28]. Members of the FOXO
forkhead transcription factor subfamily (e.g., FOXO1a and
FOXO3a) are also important substrates of AKT. Mice that lack
Foxo3a display follicular activation and depletion of oocytes
with secondary infertility, which suggests an important role for
this protein in ovarian function [29]. AKT also phosphorylates
GSK3B, which may be involved in the regulation of ovarian
gene expression by FSH [21, 30]. Therefore, the major roles of
FOXO and GSK3B as downstream effectors of AKT signaling
involve the co-ordination of cell proliferation and differentiation in the ovary, and defects in these events in Irs2 null ovaries
are likely to account for many of the observed abnormalities.
The AKT/forkhead signaling cassette has been demonstrated to
regulate directly cyclin D2 function in rat granulosa cells, and
GSK3B has also been shown to regulate D-type cyclin function
1052
NEGANOVA ET AL.
FIG. 6. Cyclin D2 and CDKN1B immunolocalization in ovaries from WT and Irs2 null mice. A–E) Immunostaining for cyclin D2 in WT ovaries.
Immunostaining for cyclin D2 is present in the primary and secondary follicles (A), in granulosa cells of transitional follicles (B), in the cumulus and mural
granulosa cells (C) 46 h after eCG treatment, and 8.5 h after superovulation induction in cumulus cells and mural granulosa cells (D and E). F–J)
Immunostaining for cyclin D2 in Irs2 null ovaries. Immunostaining for cyclin D2 is detected in the primary and secondary follicles (F), in transitional
follicles (G), 46 h after eCG in a small antral follicle (H), and after superovulation induction (I and J). K–O) Immunostaining for CDKN1B in WT ovaries.
CDKN1B immunostaining is absent in the secondary follicle and present in the corpus luteum (CL) (K), in small growing transitional follicles (L), in large
antral follicles 46 h after eCG (M), and after superovulation induction (N and O). P–T) Immunostaining for CDKN1B of Irs2 null ovaries. Immunostaining
for CDKN1B is detected in the secondary and small transitional follicles (P), in a small antral follicle (Q), 46 h after eCG treatment (R), and 8.5 h after
superovulation induction (S and T). Bar ¼ 100 lm.
IRS2 REGULATES OVARIAN FUNCTION
[31]. AKT/forkhead signaling also regulates CDKN1B function through both transcriptional and phosphorylation events.
Deletion of cyclin D2 in mice results in impaired granulosa cell
proliferation, impaired follicular growth, trapped oocytes in the
corpora lutea, and ovulatory failure [32, 33]. Mice that lack
CDKN1B have normal follicular growth but have impaired
luteinization of granulosa cells [34]. In Irs2 null ovaries, we
have demonstrated defects in the regulation of both cyclin D2
and CDKN1B due to impaired upstream AKT signaling, which
may explain the functional abnormalities of ovarian cellular
function in these mice. Reduced AKT activity may lead to the
earlier expression of CDKN1B seen in the granulosa and
cumulus cells of Irs2 null follicles. Disordered co-ordination of
follicular and oocyte functions may also account for the
frequent observation of trapped oocytes in Irs2 null follicles.
In summary, we have shown that CNS IRS2 signaling
pathways are not required for female reproductive function. In
contrast, signaling events downstream of IRS2 in the ovary
play critical roles in follicular development and ovulation by
regulating key components of the cell cycle apparatus involved
in the co-ordination of cell proliferation and differentiation.
ACKNOWLEDGMENTS
We thank P. Smith and A. Parlow of the National Hormone and Pituitary
Program and Pituitary Hormones and Antisera Centre for reagents.
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