BACKGROUND: Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs). CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs). METHOD: In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16). RESULTS: Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++) in the oocytes of all primordial, and 57.4 ± 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+). Interestingly, primary follicles with weaker (+) oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++) oocyte staining (55.6 ± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 μm, respectively, p < 0.0001). This difference in follicle diameter was mainly due to a larger mean oocyte diameter (primary follicles, stronger vs. weaker, 19.6 ± 0.6 vs. 31.5 ± 1.4 μm, p < 0.0001). Oocytes of atretic follicles showed stronger staining with all four CDKIs. CONCLUSIONS: These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth

Bayrak, Aykut

Oktay, Kutluk

English

PubMed

Aykut Bayrak

Kutluk Oktay

Springer - Publisher Connector

BioMed Central
Reproductive Biology and 
Endocrinology
ssOpen AcceResearch
The expression of cyclin-dependent kinase inhibitors p15, p16, p21, 
and p27 during ovarian follicle growth initiation in the mouse
Aykut Bayrak1,2 and Kutluk Oktay*1,2,3
Address: 1Department of Anatomy & Cell Biology, State University of New York Health Science Center, Brooklyn, NY, USA, 2Department of 
Obstetrics & Gynecology, New York Methodist Hospital, Brooklyn, NY, USA and 3Center for Reproductive Medicine & Infertility, Weill Medical 
College of Cornell University, New York, NY, USA
Email: Aykut Bayrak - bayrak@usc.edu; Kutluk Oktay* - kuo9001@med.cornell.edu
* Corresponding author    
Abstract
Background: Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs).
CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs).
Method: In this study we tested the expression of CDKIs p15, p16, p21 and p27 by
immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle
growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16).
Results: Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle
stage. However, p16 staining was stronger (++) in the oocytes of all primordial, and 57.4 ± 3.1% of
primary follicles compared to the remaining primary and more advanced follicles (+). Interestingly,
primary follicles with weaker (+) oocyte staining for p16 had significantly larger mean follicle
diameter compared to the primary and primordial follicles with stronger (++) oocyte staining (55.6
± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 µm, respectively, p < 0.0001). This difference in follicle diameter
was mainly due to a larger mean oocyte diameter (primary follicles, stronger vs. weaker, 19.6 ± 0.6
vs. 31.5 ± 1.4 µm, p < 0.0001). Oocytes of atretic follicles showed stronger staining with all four
CDKIs.
Conclusions: These preliminary findings suggest that the initiation of oocyte growth, which seems
to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further
studies are needed to investigate the factors that regulate the expression of p16 in the oocyte,
which might also govern the initiation of primordial follicle growth.
Introduction
The onset of growth of an individual primordial follicle is
unpredictable; some beginning shortly after formation
while others may remain "quiescent" for many years. By
which mechanism the primordial follicles are selected to
grow is unknown. We previously showed that the Prolif-
erating Cell Nuclear Antigen (PCNA) is expressed in the
rodent ovarian follicles at the earliest sign of growth [1].
PCNA is a co-factor of cyclin-D and it makes a complex
with cyclin-D, a cyclin dependent kinase (CDK), and a cy-
clin dependent kinase inhibitor (CDKI). The progression
of cells through the cell cycle is regulated by a family of
protein kinases known as the cyclin-dependent kinases
(CDKs). The sequential activation of the members of this
family and their phosphorylation of certain substrates
promotes the progression through the cell cycle. Cyclins
Published: 7 May 2003
Reproductive Biology and Endocrinology 2003, 1:41
Received: 25 February 2003
Accepted: 7 May 2003
This article is available from: http://www.RBEj.com/content/1/1/41
© 2003 Bayrak and Oktay; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in 
all media for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 5
(page number not for citation purposes)
Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/41function as the positive regulators of CDKs. D-type and E-
type cyclins assemble with CDKs during the G1 phase and
these holoenzymes act as rate-limiting controllers to regu-
late passage through the restriction point and the subse-
quent onset of DNA replication [2,3]. D-type cyclins are
usually synthesized by mid-G1 phase and accumulate to a
maximum as cells advance through the G1/S boundary.
D-type cyclins act as regulatory subunits for two cyclin-de-
pendent kinases CDK4 and CDK6 [4,5]. The synthesis of
another G1 phase cyclin, cyclin E, increases in late G1 and
decreases once DNA replication is initiated. Cyclin E
forms complexes during this interval with CDK2. Cyclins
and CDKs assemble into complexes with one another as
cells progress through G1 phase, cyclins being required to
activate the serine-threonine kinase activity of their cata-
lytic partners. Furthermore, CDK-activating kinase (CAK)
phosphorylates cyclin-bound CDKs on a single threonine
residue, a modification that is essential for their activity
[6–9].
Cyclin-dependent kinase inhibitors (CDKIs) are proteins
that bind to and inhibit the activity of CDKs. Two major
classes of CDK inhibitors have been identified. The p16
family (p15, p16, p18 and p19) binds to and inhibits the
activities of CDK4 and CDK6. The p21 family (p21, p27,
p28 and p57) can bind to broad range of CDK-cyclin com-
plexes and inhibit their activities. CDKIs are capable of
suppressing growth, and several lines of evidence strongly
suggest that at least some CDKIs may be tumor suppressor
proteins [10,11].
In this study, we studied the expression of four CDKIs;
p15, p16, p21 and p27 in mouse ovaries by immunohis-
tochemistry to assess whether the initiation of primordial
follicle growth was associated with the expression of
CDKIs.
Materials and Methods
The study was approved by the institutional animal care
committee at the State University of New York Health Sci-
ence Center at Brooklyn.
Tissue Preparation and Immunohistochemistry
Ovaries were collected from 60-day-old cycling B6D2F1/J
mice (n = 16) during estrus. Ovaries were fixed in Bouin's
solution for 6 hours and transferred into 70% alcohol and
incubated for 24 hours at room temperature. After 24
hours, ovaries were embedded in paraffin and serially sec-
tioned at 5 µm. Sections were mounted on coated slides
(Vectabond; Vector Laboratories, Burlingame, CA).
Forty sections were randomly selected from one ovary in
each animal and stained for CDKIs p15, p16, p21 and p27
(Vector Laboratories, Burlingame, CA). Sections were de-
paraffinized in xylene for 10 min, gradually rehydrated in
ethanol for 9 min. After washes with water and TBS either
normal goat serum for p16 and p27 (rabbit polyclonal an-
tibodies) or normal rabbit serum for p15 and p21 (goat
polyclonal antibodies) were added for 20 min at 37°C to
prevent non-specific binding. Then first antibodies were
added (1/50) for p15, p21, p27 and (1/100) for p16 in
TBS and sections were incubated for 90 min at 37°C. After
a wash in TBS, sections were quenched in 3% hydrogen
peroxidase to block endogenous peroxidase activity. Then
sections were immersed in water and TBS and second an-
tibodies were added at a dilution of 1/100 for 30 min at
37°C. Anti-rabbit antibody for p16 and p27 (rabbit poly-
clonal antibodies) and anti-goat antibody for p15 and
p21 (goat polyclonal antibodies) were used for second an-
tibodies. After a wash in TBS, avidin-biotin complex was
added for 20 min at 37°C followed by diaminobenzadine
(DAB) solution for 4 min. Sections were then counter-
stained with hematoxylin. The immunostaining intensity
was quasi-quantified using a "-" to "+++" scale. This was a
scale comparing relative staining intensity within each an-
tibody amongst cell types and follicle stages.
Morphometry
All sections were examined under 100× to 1000× magni-
fication under light microscopy to determine immunos-
taining characteristics and the follicular stages. Earlier
stages of follicles were classified as described previously
[1]. Primordial follicle, an ungrown oocyte encapsulated
by flattened or squamous cell; and primary follicle, with a
single layer of cuboidal granulosa cells.
One section from each p16-stained slide was randomly se-
lected for oocyte and follicle measurements. There were 2
sections per animal for a total of 32 sections. All primor-
dial (n = 249) and primary follicles (n = 160) with the
oocytes that had a nucleus were examined in each section.
Oocyte and follicle diameters were measured by a mi-
crometer. Mean oocyte and follicle diameters in each fol-
licle category were compared in relation to the intensity of
the staining for p16.
Statistical Analysis
Statistical analysis was performed using a two-way analy-
sis of variance. Statistical significance was set at p = 0.05.
Results
Quasiquantification of immunostaining intensities for
each CDKI is shown in table 1. Expression of p15, p16,
p21 and p27 did not vary in granulosa and theca cells by
the follicle stage. However, p16 staining was stronger (++)
in the oocytes of all primordial (Figure 1A and 1E) and
57.4 ± 3.1% of primary follicles (Figure 1B,1C) compared
to the remaining primary (Figure 1D,1E) and more ad-
vanced follicles (+) (Figure 2A,2B). Interestingly, primary
follicles with weaker (+) oocyte staining for p16 (FigurePage 2 of 5
(page number not for citation purposes)
Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/411D,1E) had significantly larger mean follicle diameter
compared to the primary and primordial follicles with
stronger (++) stained oocytes (55.6 ± 2.1 vs. 32.0 ± 1.0
and 26.5 ± 0.7 µm, respectively, p < 0.0001). This differ-
ence in follicle diameter was mainly due to a larger mean
oocyte diameter (primary follicles, stronger vs. weaker,
19.6 ± 0.6 vs. 31.5 ± 1.4 µm, p < 0.0001). The oocytes of
atretic follicles showed stronger staining with p16 (Figure
2C) as well as other CDKIs (not shown).
Discussion
Here we showed that p16 is strongly expressed in the
oocytes of primordial and early primary follicles (primary
follicles with "ungrown" oocytes) in comparison to pri-
mary follicles. The change in the expression of p16 ap-
pears to coincide with the first measurable sign of oocyte
growth. These findings suggest that the initiation of
oocyte growth, which seems to lead follicle growth, is as-
sociated with diminished p16 expression in the mouse
ovary.
Oocyte in adult mouse ovary has entered the first meiotic
prophase and arrested in early M-phase. Oocyte in adult
mice has already finished DNA replication (S-phase). It is,
therefore curious that p16, a factor controlling G1/S, plays
a role in healthy oocyte. However recent studies have
shown that TGF-B family member ligands such as GDF-9,
which influence cell proliferation and differentiation, are
Figure 1
Changing expression of p16 in oocytes of follicles initiating growth. Original magnification 1000X. (A)Increased expression of 
p16 in primordial follicle oocytes (arrows); (B&C) Increased expression of p16 in primary follicles with unenlarged oocytes 
(arrows); (D) Diminished expression of p16 after enlargement of the oocyte in primary follicles. (E) Note the primordial follicle 
(arrow) with significantly higher expression of p16 in its oocyte compared to the adjacent primary follicle with an enlarged 
oocyte. Bar = 50 µm.Page 3 of 5
(page number not for citation purposes)
Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/41involved in follicle growth initiation and oocyte growth
[12,13]. Interestingly, other studies have shown that TGF-
B's antiproliferative effects on some cell types is mediated
by the p16 family CDKIs [14]. Since GDF-9 is only synthe-
sized in the oocyte, and since it is not expressed in the
oocyte until primary follicle stage, a different member of
the TGF-B family such as Activin-A [15] may be influenc-
ing oocyte p16 expression via paracrine mechanisms.
Figure 2
p16 expression in later stage follicles. Original magnification 200X. (A) multiple preantral follicles with diminished p16 staining; 
(B) an antral follicle with diminished p16 staining; (C) an atretic follicle with increased p16 staining in the oocyte. Bar = 100 µm.
Table 1: Quasiquantification of the expression of CDKI in ovarian follicular cells*
Cell Type p15 p16 p21 p27
Granulosa -/+ -/+ -/+ -/+
Oocyte + +/++ -/+ -/+
Theca + +++ -/+ -/+
Luteal + ++ +/++ -/+
*This is a relative scale within each antibody group.Page 4 of 5
(page number not for citation purposes)
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Role of p16 in primordial follicle quiescence might be
through an entirely different mechanism. CDKIs also play
role in contact inhibition [16]; and thus high expression
of p16 in ungrown oocytes may also reflect inhibition be-
stowed on them by pregranulosa cells. It is therefore plau-
sible that p16's role in oocyte growth may be different
than its general role in cell cycle control.
We also found that atretic follicles strongly expressed all
four CDKIs tested. This finding is consistent with the pre-
vious work in other cell types which indicated the involve-
ment of CDKIs in apoptosis [17]. P16 expression was also
high in theca cells regardless of follicle stage. This is con-
sistent with the low mitotic activity of these cells.
In addition, granulosa cells of healthy growing follicles
showed weaker staining. This finding complements the
finding from our previous study that PCNA is strongly ex-
pressed in granulosa cells starting from the primary folli-
cle stage [1]. PCNA is a cofactor of Cyclin D, and p16
family of kinase inhibitors inhibit the activity of CDKs as-
sociated with this cyclin. Downregulation of p16 would
result in activation of this CDK and increased PCNA and
Cyclin D expression, which result in the progression of
cell cycle through the G1/S phase. There is also an inverse
relationship between PCNA and p16 expression in oocyte
growth. PCNA is expressed after the oocyte enlargement
in primary follicles, exactly the same stage when p16 ex-
pression disappears.
Factors regulating the expression of p16 are unknown. Re-
cent work has shown that p16 expression may be regulat-
ed by Jun family of transcription factors. Interestingly, jun
family of transcription factors play an important role in
cell proliferation [18]. These transcription factors are acti-
vated by the enzyme Jun amino terminal kinase (JNK), the
expression of which can be regulated by UV, genotoxic
stress, and cytokines. Our current research focuses on
these and other potential upstream regulators of p16.
In conclusion, this preliminary study suggests that quies-
cence of primordial follicles might be maintained by high
expression of p16. Cyclin dependent kinase inhibitors
have been shown to be growth suppressors, and the ab-
sence or decreased expression of p16 strongly coincides
with the first measurable follicle growth. Further studies
will be helpful in determining the factors that regulate the
expression of p16, which might also govern the initiation
of follicular growth.
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The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse

Abstract Background Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs). CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs). Method In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16). Results Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++) in the oocytes of all primordial, and 57.4 ± 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+). Interestingly, primary follicles with weaker (+) oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++) oocyte staining (55.6 ± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 μm, respectively, p  Conclusions These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth.</p

Bayrak Aykut

Oktay Kutluk

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The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse

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