MINI REVIEW

Characterization of Two FSH Inducible Genes,
a Nuclear Transcriptional Co-activator p120
and a Novel Serine Protease-like Protein,in the Rat Ovarian Granulosa Cells
Miki YOSHINO1,2), Tetsuya MIZUTANI1,2), Kazuya YAMADA1,2), Takeshi ARIMA1,2), Takashi YAZAWA1,2),
Hiroko OGATA-KAWATA1,2)), Toshio SEKIGUCHI1,2)), Takashi KAJITANI1,2) and Kaoru MIYAMOTO1,2)
１）Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan
２）CREST, JST (Japan Science and Technology), Japan

Abstract

FSH. In this report, characterization of the co-activator p120 and the novel serine protease-like protein in

Ovarian follicular development is primarily depen-

the ovary will be described.

dent on pituitary gonadotropins. We report here theidentification of two genes, a nuclear transcription co-

Subtraction cloning and DNA microarray

activator p120, and a novel serine protease-like protein, as FSH inducible genes in the rat granulosa cells.

The subtraction cloning and DNA microarray tech-Characterization of the genes revealed that rat p120

nology are quite powerful methods for the comprehen-

may work together with PR in the granulosa cells of

sive analysis of gene expression. In order to identify

large antral follicles to promote the ovulation events,

FSH-inducible genes in the rat granulosa cells, sub-and that the novel serine protease-like protein may be

traction cloning and DNA microarray analyses wereinvolved in the proteolytic process during the follicularperformed. Cultured granulosa cells (5.0×106 viable

development and ovulation process.

cells) were treated with or without 30 ng/ml FSH for
15 hours. Messenger RNAs from FSH treated cells as

Introduction

well as control cells were converted to double stranded cDNAs, and subtraction cloning was performed as

Follicular growth is primarily controlled by pituitarypreviously reported [2]. Resulting cDNAs of size-range

gonadotropins, LH and FSH. Especially FSH stimulates

between 0.5-2.0 kbp long were subcloned to constructproliferation and differentiation of granulosa cells ofa subtraction plasmid cDNA library. About 400 clones

small follicles in the ovary, and promotes their develop-

in the plasmid library were randomly picked up andment to pre-ovulatory follicles [1]. Recently we identi-

their nucleotide sequences were partially determined.

fied many gonadotropin inducible genes expressed in

For DNA microarray analysis, mRNAs from the cells

the ovary by subtraction cloning and DNA microarray

treated with or without FSH were labeled with fluo-

analyses, and found that a transcriptional co-activator

rescent dyes, Cye3- or Cye5, respectively. The labeled

p120 and a novel serine protease-like protein were

cDNA pools were hybridized to microarray glass slides

strongly induced in the rat cultured granulosa cells bycontaining 3200 cDNA elements. As listed in Table 1,
hundreds of candidate genes were picked-up and 10

Correspondence：Kaoru MIYAMOTO, Department ofBiochemistry, Faculty of Medical Sciences, University of
Fukui, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan
TEL: +81-776-61-8316
FAX: +81-776-61-8102
E-mail: kmiyamot@fmsrsa.fukui-med.ac.jp

genes were confirmed as FSH inducible genes by thesemi-quantitative RT-PCR analysis. Among these
FSH-inducible genes, we describe here the expression
and localization of the transcriptional co-activator
p120, as well as those of the novel serine protease-like

Japanese Journal of Reproductive Endocrinology（2004）9：29-34

29Miki YOSHINO et al.

Table 1. Induced genes in the rat granulosa cells stimulated with FSH
for 15 hours
Accession
numberCharacterization of rat p120, an FSH inducible
transcriptional co-activator

Definition

In this study we identify rat homologue of p120, a

AB180485

Human thyroid hormone receptor coactivating protein
like protein (rat p120)

AB180912

Unknown serine protease like protein

NM̲017290

Rat ATPase, Ca2+ transporting, cardiac muscle, slow
twitch 2 (Atp2a2)

NM̲022265

Rat programmed cell death 4 (Pdcd4)

M64393Rat 3-α-hydroxysteroid dehydrogenase(3-α-HSD)

NM̲019683

Mouse globin inducing factor, fetal (Gbif-pending)compared with that of human p120. It contains pro-NM̲011462

Mouse spindlin (Spin)line-rich, acidic, and bromo-domains, respectively, andU23776Rat Eker rat-associated intracisternal-A particle
element

there are two LXXLL-motifs between proline-rich andNM̲012600

Rat Malic enzyme 1, soluble (Me1)

AY009092

Rat retrovirus SC1

nuclear transcription co-activator, as one of the FSH
inducible genes in the rat granulosa cells. We thencloned a full-length cDNA encoding rat p120 and thenucleotide sequence was determined. Fig. 1 shows the
deduced amino acid sequence of rat p120, which isacidic domains. The sequences are well conserved between rat and human (94% identity at amino acid level), except that rat p120 has 45 amino acid residues
extended at N-terminus. The transcriptional co-activa-

protein in the ovary during the follicular development

tor p120 was originally isolated as a novel nuclear co-and the ovulation processes.

activator for thyroid hormone receptor [3]. Further
studies revealed that p120 also interacts with androgen receptor or 9-cis-retinoic acid receptor (RXR)

Fig. 1. Deduced amino acid sequence of rat p120
The proline-rich domain between 223 and 288 amino acid residues from the N-terminus is underlined, and acidic amino acid-rich domain
between 602 and 731 is shown by bold type. The bromodomain between 809 and 894 is boxed. The putative NR box (LXXLL motif), which
is necessary for the interaction with nuclear hormone receptors, is boxed by colored background. Since there are high homology between rat
and human p120 sequence (94% identity at amino acid level), rat p120 is expected to have similar functions.

30

Japanese Journal of Reproductive Endocrinology Vol. 9 2004

Rat p120 and a Novel Serine Protease in the Ovary

functioning as a co-activator of these nuclear receptors [4]. The receptors belong to the nuclear receptor
super-family proteins that activate the transcription of
their target genes when the steroid hormones or ligands bind to them. The nuclear hormone receptors
generally bind to the respective hormone responsive
elements (HREs) in the upstream regions of the target genes, and activate the transcription. Deficienciesof estrogen receptors or progesterone receptors (PR)result in various defects of reproductive functions,such as abnormal follicular development, anovulation,and infertility [5-7]. Transcriptional co-activators are
required to form active protein complexes with thehormone nuclear receptors and the basic transcription
machinery proteins for the target gene transcription.
Co-activators p160/SRC and CBP/p300 are known tointeract with the nuclear hormone receptors at the
C-terminal AF-2 region through the LXXLL-motif(NR box) [8-10]. Knock-out mice of steroid receptor
co-activator 3 (SRC-3), a member of p160 family, exhibit infertility [11], and those of nuclear receptor interacting protein 1 (Nrip1/RIP140) are also infertile due

Fig. 2. Localization and transcriptional co-activator function of rat
p120(A) Sections of ovaries from immature rats primed with hCG for
4 hours followed PMSG 48 hours were hybridized with p120
(a, b; antisense probe, c; sense probe) or PR (d, e; antisense
probe, f; sense probe). Expression of these genes was completely co-localized in the granulosa cells of large antral follicles, suggesting that p120 works together with PR in the ovary.
(B) Transcriptional co-activator activity of rat p120 was examined by co-transfection experiments. CV-1 cells were transfected with a luciferase reporter plasmid and the human PR expression vector with or without 10 -7M progesterone, in thepresence or absence of p120. The luciferase activity of PR
was upregulated about two fold in the presence of p120.

to the blockage of the oocytes release from the Graafian follicles by the ovulatory stimuli [12]. These reports
indicate that co-activators are also important for theshown in Fig. 2B, p120 enhanced the transcription ac-follicular development and ovulation. Therefore we ex-tivity of PR in the presence of the ligand progesterone.

amined expression and localization of p120 gene in the

These observations suggest that p120 may work to-ovary during the follicular development using imma-

gether with PR in the granulosa cells of ovulatory folli-

ture rats primed with gonadotropins. As shown in Fig.

cles to promote the ovulation in the ovary.

2A, in situ hybridization revealed that the induction
pattern of p120 was very similar to that of PR in the
granulosa cells of large pre-ovulatory follicles. It is

A novel serine protease-like protein in the ovary

well established that PR participates in the ovulation
process occurring in the pre-ovulatory large antral fol-

We also examined expression and localization of thelicles. Ovulation is triggered by LH surge from the pi-

novel FSH-inducible serine protease-like protein intuitary. About 4 hours after the LH surge, expression

the ovary during the follicular development, in orderof PR is rapidly induced in the granulosa cells of pre-to check possible involvement of the gene in someovulatory large antral follicles destined to ovulate [13,ovarian proteolytic processes. The extracellular matrix14]. Progesterone binds to the induced receptor to acti-(ECM) influences basic cellular processes such as pro-vate gene expression of specific proteases, ADAMTS-1

liferation, differentiation, migration and adhesion, andor Cathepsin L, which are thought to work on follicle

is fundamental to normal development. A tightly regu-

rupture by degrading the ovulating follicle walls [15].

lated proteolytic activity controls the remodeling and

In addition, we examined co-activator functions of rat

degradation of matrix components in these processes.

p120 by co-transfection experiments to show that p120

In the ovary, many physiological and pathological pro-may promote the action of PR during the ovulation. As

cesses including follicular development, ovulation and

MINI REVIEW

31

Miki YOSHINO et al.

angiogenesis require a regulated turnover of ECM

residues with a putative signal sequence, and putative

components. Although a lot of proteolytic enzymes in-active site Asp, His, and Ser residues that are con-cluding members of the plasminogen activator (PA)served in serine protease family such as PA protein.

or matrix metalloproteinase (MMP) have been recog-

As shown in Fig. 4, RT-PCR revealed that the serine

nized to be involved in several ovarian proteolytic

protease was expressed in the brain (lane 1), adrenal

events, a definitive functional role for individual prote-(lane 4), intestine (lane 9), testis (lane 6), and ovary

ases has yet to be demonstrated [16]. Indeed, there ex-(lane 11). PMSG treatment increased the gene expres-ist many deficient mouse strains that lack individualsion in the ovary after 48 hours (lane 12).

matrix degrading proteases, but these mice are fertile

Northern blot analysis revealed that the serine pro-and don't have major defects of ovulation and female

tease mRNA level was rapidly induced by the FSH

reproduction, as shown in knockout mice of PA [17].

treatment within 3 hours, and the levels were kept

In this context, we cloned a full-length cDNA encod-

high throughout the treatment (Fig. 5A). As shown ining a novel FSH inducible ovarian serine protease-like

Fig. 5B, the treatment of immature rats with PMSG inprotein and the nucleotide sequence was determined.
Fig. 3 shows the nucleotide sequence of the serine protease-like protein along with deduced amino acid sequence. The gene encodes a protein of 406 amino acid

Fig. 4. Expression of the serine protease-like protein gene in various
tissues
RT-PCR was performed using various tissues. The samples arebrain (lane 1), pituitary (lane 2), liver (lane 3), adrenal (lane 4),
uterus (lane 5), testis (lane 6), kidney (lane 7), spleen (lane 8),
intestine (lane 9), stomach (lane 10), untreated ovary (lane 11),
ovary treated with PMSG for 48 hours (lane 12).

Fig. 3. Nucleotide and deduced amino acid sequence of the serine
protease-like protein
The amino acids are numbered starting from the initiation codon. The deduced signal sequence between 1 and 17 residues
from the N-terminus is underlined, and conserved residues between 162 and 167 involving the putative active site histidine
residue is boxed.

32Japanese Journal of Reproductive Endocrinology Vol. 9 2004

Fig. 5. Induction of the serine protease-like protein gene expression in
the ovaryNorthern blot analysis of the serine protease-like protein mRNAin cultured granulosa cells treated with FSH (A), in immature
rat ovaries primed with PMSG (B), and in immature rat ovaries
primed with PMSG followed by hCG (C).

Rat p120 and a Novel Serine Protease in the Ovary

vivo also induced the protease gene expression in the

Localization of the serine protease in the ovary wasovary within 6 hours after the treatment, with a tran-also examined by in situ hybridization using adjacentsient peak at 24 hours after the treatment. Further

frozen sections as shown in Fig. 6. Immature female

hCG treatment also increased the gene expression

rats were treated either with PMSG or PMSG/hCG.

with a peak at 2 hours as shown in Fig. 5C.

The expression of the gene was induced in the granulosa cells of large antral follicles 4 hours after the hCGtreatment (Fig. 6C). Since the treatment describedabove was typical for ovulation induction, the expression patterns of this serine protease-like protein suggest possible involvement of this protein in the ovulatory events. Expression of the serine protease wasconfirmed in transiently transfected CHO cells byWestern blotting for FLAG epitope that engineered at
the C terminus (Fig. 7). The detected band was consistent with the predicted molecular mass of 55 KDa.
Further studies will be required to show the substratespecificity of this protein in order to clarify its physiological role during the follicle development. It is wellknown that ADAMTS-1 is multifunctional MMP and

Fig.6. In situ hybridization of the serine protease-like protein
Ovaries from 21-d-old immature rats were used. (A) Control
ovaries were hybridized with the antisense probe of the serine
protease-like protein (a, b), (B) those treated with PMSG for 24hours were hybridized with the antisense probe (c, d), or hybridized with sense probes (e, f), (C) those treated with hCG for 4
hours after the PMSG treatment were hybridized with the antisense probe (g, h). Original magnification was x100. Scalebars, 0.1 mm.

induced by LH surge in the granulosa cells and cumulus cells, and has a critical downstream role in mediating the PR-regulated ovarian activity that culminatesin the rupture of a follicle [15]. The novel serine protease-like protein described here may also be a memberof crucial proteases involved in such processes.

Conclusion
In this report, we described the isolation and thecharacterization of two FSH inducible genes in the rat
ovarian granulosa cells. We showed here that rat p120,
a transcriptional co-activator, interacts with PR to enhance their activity, and co-localized with PR in the
granulosa cells of large antral follicles. Our observation
Fig.7. Recombinant expression of the serine protease-like protein
CHO cells were transfected with 10 μg of the serine proteaseFLAG/pcDNA3 construct or with control pcDNA3 vector. After
24 hours, culture medium were collected and concentrated.
Cell lysates were also prepared. Samples were resolved on a
10 % SDS-PAGE gel and immunoblotted with the M2 antiFLAG epitope antiserum (Sigma). Cell lysate from the serine
protease-like protein-FLAG/pcDNA3 transfected cells (lane 1),
from the control vector transfected cells (lane 2), the concentrated culture medium from the serine protease-like proteinFLAG/pcDNA3 transfected cells (lane 3), and the concentrated
culture medium from the control vector transfected cells (lane4). A specific band corresponding the recombinant protein was
detected at 55 KDa. This indicates that the novel serine protease-like protein was actually translated in the cells, and may
function as an ovarian protease.

suggests that the co-activator p120 may participate in
the ovulation processes co-working with PR. We also
identified a novel serine protease-like protein in the
rat granulosa cells. Although further studies should
clearly be needed for its role in the ovary, the novel
serine protease-like protein may be a new crucial
member of proteases involved in the proteolytic processes in the ovary.

MINI REVIEW

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Miki YOSHINO et al.

Acknowledgement
This work was supported in part by a grant from the
Smoking Research Foundation, and by 21st Century COE
Program (Medical Science).

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