ORIGINAL PAPER

Jpn J Reprod Endocrinol ⓒ2004 by
Japan Society of Reproductive Endocrinology

FSH-induced Activation of PI 3-kinase-PKB Pathway is
Essential for LH Receptor Formation in
Cumulus Cells during Meiotic Resumption of Porcine Oocytes
Masayuki SHIMADA and Yasuhisa YAMASHITA
Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan

Abstract. It has been reported that the PI 3-kinase-protein kinase B (PKB) pathway is activated by FSH stimulationand plays an important role in follicular growth and maturation. However, the critical role of the PI 3-kinase-PKB pathway in cumulus cell differentiation during in vitro maturation of porcine oocytes remains unclear. The present study
showed that both PI 3-kinase and PKB were detected by Western blotting in cumulus cells of COCs that were cultured
with or without FSH. However, positive signals against anti-phospho-PKB specific antibody, which recognized PKB
phosphorylated at Ser473, were observed when COCs were cultured with FSH for 20 hr. The addition of the PI 3-kinase inhibitor LY294002 to FSH-containing medium suppressed the phosphorylation of PKB in cumulus cells. We already reported that expression of the LH receptor (LHR) gene is one of the cumulus cell responses to FSH stimuli.
Four types of LHR mRNA were detected in FSH-stimulated cumulus cells by RT-PCR. The large form was identified
as a part of the porcine full-length LHR cDNA, which was able to bind its ligand. The level of full-length type LHRmRNA was significantly decreased by the addition of LY294002 to FSH-containing medium. These results suggest that
FSH up-regulates LHR formation through a PI 3-kinase-PKB dependent pathway in cumulus cells of COCs during invitro maturation of porcine oocytes.

Key words: PI 3-kinase, Akt/PKB, LH receptor, Cumulus cells, Pig oocytes

Introduction

Akt/protein kinase B (PKB) phosphorylation and activation
in a way that is dependent on phosphatidylinositol 3-kinase

It is well documented that FSH stimulates adenylcyclase(PI 3-kinase) in granulosa cells [6,7]. In cumulus cells, wewith a resultant increase in cAMP synthesis [1,2]; however,

have reported that PKB activity significantly decreases in asignal transduction of the cAMP-protein kinase A (PKA)

dose-dependent manner in the relation to the PI 3-kinase

pathway alone does not represent the expression pattern ofinhibitor, LY294002, when porcine cumulus-oocyte com-the many genes activated by FSH [3]. Actually, FSH stimu-

plexes (COCs) were cultured with FSH [8], suggestinglation has been shown to be mediated by several pathways,that FSH stimulation also activates the PI 3-kinase-PKB

for example the MAP kinase pathway [4] and the intracellu-

pathway in cumulus cells as well as granulosa cells.

lar Ca2+pathway [5] in addition to the cAMP-PKA pathway.

Recently, it has been reported in rat granulosa cells that

Several researchers have shown that FSH also increases

the transcriptional activity of the LH receptor (LHR) gene
was up-regulated by stimulation with FSH and that the induction was significantly suppressed by LY294002 [9]. In

Accepted: August 4, 2004
Correspondence: Masayuki SHIMADA, Department of Applied
Animal Science, Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima,739-8528, Japan
TEL: +81-82-424-7899
FAX: +81-82-424-7899
E-mail: mashimad@hiroshima-u.ac.jp

our previous study [10,11], we showed that the expression

This work was supported by the following sources of funding:
Grant-in-Aid for Scientific Research (M.S., No.14760179,
16780195) and Research Fellowship for Young Scientist (Y.Y.,
No.08254) from the Japan Society for the Promotion of Science.

from these reports including our previous study, we esti-of the LHR gene in cumulus cells of porcine COCs was upregulated by FSH and that the binding of LH to its receptor
induced progesterone production. Furthermore, high-levelprogesterone production was significantly reduced by
LY294002 during in vitro maturation of COCs [8]. Judging
mated that the PI 3-kinase-PKB pathway in cumulus cells
plays an important role in LHR formation.

Japanese Journal of Reproductive Endocrinology（2004）9：49-54

49

Masayuki SHIMADA et al.

In this study, to investigate the role of the PI 3-kinasePKB pathway in the formation of LHR in cumulus cells,３）RT-PCR for LHR mRNA

COCs were cultured with both FSH and PI 3-kinase inhibi-

Oligonucleotide primers used for amplification of thetor, LY294002 for 20 hr. The expression of the LHR gene

LHR were designed from known cDNA sequences of four

was analyzed by RT-PCR technique. The protein level of PI

porcine LHR isoforms (GenBank accession number:

3-kinase and the phosphorylated status of PKB were de-M29525) [15]. The upstream primer (5 -CCAATCTCCTA-

tected by Western blotting.

GATGCCACATTGAC-3 ) is identical to nucleotides
861-885 of the porcine cDNA, and the downstream primer

Materials and methods
１）In vitro cultivation of porcine COCs
Isolation of porcine COCs was described previously [12].

(5 -GCTCAGCAACAGAAAGAAATCCC-3 ) represents
the reverse complement of nucleotides 1959-1981. These
primer pair predicts 185-, 411-, 855- and 1121-base pair(bp) DNA fragments.

Briefly, porcine ovaries were collected from 5- to 7-month-β-actin was used as a control for reaction efficiency andold prepubertal gilts at a local slaughterhouse. Oocytes

variations in concentrations of mRNA in the original RT re-were collected from the surfaces of intact healthy antral fol-action. The β-actin primers were based on the mouse se-licles measuring from 3 to 5 mm in diameter. Oocytes hav-quences (GenBank accession number: NM009609; Tokuna-ing evenly granulated cytoplasm with at least 4 layers of

ga et al., 1986). The upstream primer (5unexpanded cumulus cells were selected and were washed-CTACAATGAGCTGCGTGTG-3 ) is identical to nucleo-

3 times with maturation medium. The COCs were cultured

tides 192-211 of the mouse cDNA, and the downstream(20/well) for 20 hr in 300 µl of the maturation medium sup-primer (5 -TAGCTCTTCTCCAGGGAGGA-3 ) represents

plemented with or without 20 ng/ml highly purified porcine

the reverse complement of nucleotides 622-641. The prim-

FSH (NIDDK, Torrance, CA, USA) at 39℃ in a humidified

er pair predicts a 450 bp DNA fragment.

incubator (95% air, 5% CO2). The maturation medium was

RT-PCR was performed according to a coupled one-step

modified NCSU37 [13] supplemented with 10% (v/v) FCS

procedure using the Access RT-PCR System (Promega)(Gibco BRL, Grand Island, NY, USA), 7 mM Taurine (Sig-with some modifications [10]. Briefly, 10 ng of total RNA

ma St Louis, MO, USA) and 4 mM hypoxanthine (Sigma).

was reverse tanscribed at 48℃ for 45 min, denatured at 94

After cultivation, COCs were collected for RNA and protein℃ for 2 min, and amplified for 26 or 32 cycles of denatur-

isolation. In the case of treatment with PI 3-kinase inhibi-

ation at 94℃ for 30s, primer annealing at 58℃ (LH recep-

tor, LY294002 (Sigma), COCs were cultured with FSH andtor) or 56℃ (β-actin) for 1 min, and extension at 68℃ for

5 µM LY294002 for 20 hr. LY294002 was dissolved in DMSO

2 min, with a final extension step of 7 min at 68℃. The am-(Sigma) at 5 mM, and stored in -30℃. The final concentra-

plified products were analyzed by electrophoresis on 2%

tion was obtained by dilution (1:1000) with the maturation

agarose gels. The intensity of the objective bands was quan-medium. Inhibitor-free medium supplemented only withtified by densitometric scanning using a Gel-Pro Analyzer

0.1% (v/v) DMSO to the maturation medium served as a(Media Cybernetics, Silver Spring, MD, USA).

control. The LY294002 concentration used herein was thesame as that used in our previous study [14].
２）RNA isolation４）Western blotting analysis
Cumulus cells were lysed in Laemmli sample buffer andprotein extracts stored at -80℃ until use. After denaturing

After cumulus cells were separated from COCs, they

by boiling for 5 min, 10 µl of each protein sample was sepa-

were washed three times in PBS. Total RNA was extracted

rated by SDS-PAGE on either 7.5 or 12.5% polyacrylamide

from cumulus cells using SV Total RNA Isolation Systemgel (Bio-Rad Laboratories, Hercules, CA), then trans-(Promega, Madison, WI, USA), according to the instruction

ferred onto PVDF membrane (Amersham Biosciences, Up-manual, and dissolved in 20 µM nuclease-free water. The

psala Sweden). The membrane was blocked with 5% (w/v)

final RNA concentrations were determined by absorbancenonfat dry milk (Amersham Biosciences) in 0.1% (v/v)

using a spectrophotometer.

Tween 20 (Sigma)/ PBS (T-PBS). Primary antibodies

50

Japanese Journal of Reproductive Endocrinology Vol. 9 2004

PI3K-PKB Acts on LHR mRNA Expression in Cumulus Cells

were added in 5% (w/v) BSA (Sigma) in PBS-T, and incubated overnight at 4℃ . Rabbit anti-phospho-PKB polyclonal antibody (Cell Signaling Technology, Inc., Beverly,MA) which recognized specifically PKB phosphorylated at
Ser473 was used at 1:2000 dilution. A rabbit polyclonal antibody against PKB (Cell Signaling Technology) was used ata dilution of 1:2000. A mouse monoclonal anti PI 3-kinase
antibody (Transduction Laboratories, Lexington, KY, USA)
which recognized the regulatory subunit of PI 3-kinase(p85α) was used at 1:500 dilution. After four washes in
T-PBS, the membranes were incubated for 1.5 hr with a
1:2000 dilution of either goat anti-rabbit IgG horseradishperoxidase-labeled antibody (Cell Signaling Technology) or
goat anti-mouse IgG horseradish-peroxidase-labeled antibody (Cell Signaling Technology) in 5% (w/v) nonfat dry
milk in PBS-T at room temperature. After five washes of
10 min each with T-PBS, peroxidase activity was visualizedusing the ECL Plus Western blotting detection system(Amersham Biosciences), according to the manufacturer s
instructions.
５）Statistical analysis
Statistical analyses of all data from three or four replicates for comparison were carried out by one-way ANOVA
followed by Duncan s multiple-range test (Statview; Abacus Concepts, Inc., Berkeley, CA).

Results and discussion
Recently, Gonzalez-Robayna et al. [6] have shown that in
granulosa cells FSH increases PKB phosphorylation and activation in a way that is cAMP-dependent and PI 3-kinasedependent. In rat Sertoli cells, the PI 3-kinase-PKB pathway has been shown to be related to FSH-regulation of cellfunctions [16]. As mentioned before, we have already demonstrated that the inhibition of PI 3-kinase activity by
LY294002 in cumulus cells interferes with gonadotropinstimulated progesterone production, and with the close ofgap junctional communications in cumulus cells during mei-

Fig. 1 The effects of LY294002 on the phosphorylation of PKB in cumulus cells of COCs cultured for 20 hr
A. Western blotting analysis of total PKB in cumulus cells of
COCs. After COCs were cultured for 20 hr, COCs were collected for protein. Total PKB was detected by a rabbit anti-PKB
polyclonal antibody which recognized both phosphorylated PKB
and non-phosphorylated PKB. The positions of molecularweight markers (in kDa) are indicated on the left. C (control);
COCs were cultured without FSH, FSH; COCs were cultured
with 20 ng/ml FSH, LY; COCs were cultured with FSH and 5µM LY294002.
B．Western blotting analysis of phosphorylated PKB in cumulus
cells of COCs. Phosphorylated PKB at Ser473 was detected byanti-phospho-PKB polyclonal antibody. The positions of molecular weight markers (in kDa) are indicated on the left. C(control); COCs were cultured without FSH, FSH; COCs were
cultured with 20 ng/ml FSH, LY; COCs were cultured with FSH
and 5 µM LY294002.
C．Western blotting analysis of PI 3-kinase in cumulus cells of
COCs. Regulatory subunit of PI 3-kinase (p85α) was detectedby mouse anti-PI 3-kinase monoclonal antibody. The positionsof molecular weight markers (in kDa) are indicated on the left.
C (control); COCs were cultured without FSH, FSH; COCs
were cultured with 20 ng/ml FSH, LY; COCs were cultured
with FSH and 5 µM LY294002.

otic resumption of porcine oocytes [8,14]. However, there islittle information about the regulation of the PI 3-kinasePKB pathway and the roles of this pathway in FSH-regula-ting analysis when COCs were cultured for 20 hr withouttion of cumulus cell function during meiotic resumption of

FSH stimulation (Fig. 1 A,C). The bands were also ob-

oocytes. The present study showed that both PI 3-kinase

served in cumulus cells of COCs that were cultured withand PKB were detected in cumulus cells by Western blot-

FSH (Fig. 1 A,C). However, the phosphorylated status of

ORIGINAL PAPER

51

Masayuki SHIMADA et al.

PKB in cumulus cells was up-regulated by culture with
FSH for 20 hr (Fig. 1 B), suggesting that the stimulation
with FSH increased the activity of PI 3-kinase-dependent
pathway in cumulus cells as well as granulosa cells.
In early antral follicles, FSH has been shown to be a major survival factor through the generation of cAMP [17].
Wang et al. [18] reported that FSH was involved in an apoptosis suppressor for granulosa cells through activation of
the PI 3-kinase-PKB pathway. In our previous study [8],few TUNEL-positive signals were detected in COCs cultured with FSH and 5 µM of PI 3-kinase inhibitor,
LY294002, whereas a high concentration of this drug (25µM) increased apoptosis of cumulus cells. However, the inhibitory effects of the addition of LY294002 to FSH-containing medium on progesterone production by COCs and
GVBD in oocytes reached a maximum at 5 µM [8]. In thepresent study, when COCs were cultured with FSH and 5µM LY294002, the intensity of the phosphorylated status of
PKB was markedly reduced and the level was comparablewith that in cumulus cells of COCs cultured without FSH(Fig. 1 B). Judging from these results, FSH-induced PKB
phosphorylation via the PI 3-kinase dependent pathway
plays an important role in the responses of cumulus cells to
FSH stimuli.
Zeleznik et al. [7] revealed that the overexpression of
dominant-negative PKB resulted in a decrease of the differentiation-associated mRNA, LHR and aromatase mRNA in
FSH-stimulated granulosa cells. We have reported by RTPCR that at least three splice variants of LHR (LHR2: 855;
LHR3: 411; LHR4: 185 bp) were detected in cumulus cells,in addition to the large form (LHR1: 1121 bp) [10,11]. The
large form (LHR1) was identified as a part of the porcine
full-length LHR cDNA (DDBJ/EMBL/GeneBank accession
number; M29525), using the Basic Local Alignment Search
Tool (BLAST, DDBJ) [11]. Loosfelt et al. [15] reported thatwhen the cDNA containing the full-length open reading
frame of LHR was transfected into COS-7 cells, hCG boundto the receptors. In the present study, the full length type of
LHR mRNA was expressed in cumulus cells of COCs that

Fig. 2. Effects of LY294002 on the expression of LHR gene in cumulus
cells of COCs cultured for 20 hr
After COCs were cultured for 20 hr, COCs were collected for
RNA, and LHR mRNA was detected by RT-PCR. The intensityof the amplified bands was quantified by densitometric scanning. The respective values of LHR were normalized according
to those of β -actin to evaluate arbitrary units of the relative
abundance of the targets. *; The addition of FSH significantly
increased the level of full length of LHR mRNA (LHR1)(P<0.01). **; The addition of LY294002 to FSH-containing medium significantly affected the level of full length of LHR mRNA
(LHR1) (P<0.01). C (control); COCs were cultured without
FSH, FSH; COCs were cultured with 20 ng/ml FSH, LY; COCs
were cultured with FSH and 5 µM LY294002.

were cultured for 20 hr with FSH (Fig. 2). The level was
significantly higher than that in cumulus cells of COCs cultured without FSH (Fig. 2). The increase of the full length

regulates the level of LHR mRNA in cumulus cells whentype LHR mRNA level in cumulus cells was significantly

COCs are cultured with FSH.

diminished by the addition of LY294002 to FSH-containing

We also reported that the binding of LH to LHR on cu-

medium (Fig. 2). Thus, the PI 3-kinase-PKB pathway up-

mulus cells significantly increased the level of cAMP in cu-

52Japanese Journal of Reproductive Endocrinology Vol. 9 2004

PI3K-PKB Acts on LHR mRNA Expression in Cumulus Cells

mulus cells of COCs [11]. The elevation of cAMP in cumulus cells stimulated progesterone production by cumulus
cells, then both decreased the proliferative activity of cumulus cells and induced differentiation, such as close of gap
junctional communication and cumulus cell expansion[19,20]. Moreover, the high concentration of progesterone
secreted by COCs improved the rate of early embryonic development to the blastocyst stage after in vitro fertilization[21]. Therefore, the FSH-induced LHR formation via the PI
3-kinase-PKB dependent pathway in cumulus cells, is very
important in the production of in vitro matured porcine oocytes with a high developmental competence.
In summary, the present study shows that the phosphorylation of PKB in cumulus cells is induced by stimulation
with FSH. When the phosphorylation of PKB was suppressed by a PI 3-kinase inhibitor, LY294002, FSH-induced
LHR mRNA expression was significantly decreased in cumulus cells of COCs. These results suggest that FSH upregulates LHR formation through the PI 3-kinase-PKB dependent pathway in cumulus cells of COCs during in vitro
maturation of porcine oocytes.

Acknowledgement
Porcine FSH was kindly provided by Dr. A.F. Parlow, the National Hormone and Pituitary Program, the National Institute of
Diabetes and Digestive and Kidney Disease, USA. Thanks to Dr.
M. Nishibori, Department of Applied Animal Science, Hiroshima
University, for technical advice on RT-PCR analysis. We thankDr. J. Ito, Mr. T. Okazaki and Mr. K. Kawahata for technical assistance, and the staff of the Meat Inspection Office in Hiroshima
City for supplying the porcine ovaries.

Reference
1. Hunzicker-Dunn M, Birnbaumer L (1976) Adenylyl cyclase activities in ovarian tissues. II. Regulation of responsiveness to LH,
FSH, and PGE1 in the rabbit. Endocrinology 99, 185-197.
2. Zeleznik AJ, Keyes PL, Menon KM, Midgley AR Jr, Reichert LE Jr(1977) Development-dependent responses of ovarian follicles to
FSH and hCG. Am J Physiol 233, 229-234.
3. Conti M (2002) Specificity of the cyclic adenosine 3 ,5 -monophosphate signal in granulosa cell function. Biol Reprod 67,
1653-1661.
4. Cameron MR, Foster JS, Bukovsky A, Wimalasena J (1996) Activation of mitogen-activated protein kinases by gonadotropins andcyclic adenosine 5 -monophosphates in porcine granulosa cells.
Biol Reprod 55, 111-119.
5. Flores JA, Leong DA, Veldhuis JD (1992) Is the calcium signal in-

duced by follicle-stimulating hormone in swine granulosa cells
mediated by adenosine cyclic 3 ,5 -monophosphate-dependent
protein kinase? Endocrinology 130, 1862-1866.
6. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL,
Richards JS (2000) Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/
Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa
cells. Mol Endocrinol 14, 1283-1300.
7. Zeleznik AJ, Saxena D, Little-Ihrig L (2003) Protein kinase B isobligatory for follicle-stimulating hormone-induced granulosa celldifferentiation. Endocrinology 144, 3985-3994.
8. Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N (2003) Phosphatidylinositol 3-kinase in cumulus cells is responsible for bothsuppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol 179,
25-34.
9. Alam H, Maizels ET, Park Y, Ghaey S, Feiger ZJ, Chandel NS, Hunzicker-Dunn M (2004) Follicle-stimulating hormone activation of
hypoxia-inducible factor-1 by the phosphatidylinositol 3-kinase/
AKT/Ras homolog enriched in brain (Rheb)/mammalian target of
rapamycin (mTOR) pathway is necessary for induction of selectprotein markers of follicular differentiation. J Biol Chem 279,
19431-19440.
10. Shimada M, Nishibori M, Isobe N, Kawano N, Terada T (2003) Luteinizing hormone receptor formation in cumulus cells surrounding
porcine oocytes and its role during meiotic maturation of porcine
oocytes. Biol Reprod 8, 1142-1149.
11. Okazaki T, Nishibori M, Yamashita Y, Shimada M (2003) LH reduces proliferative activity of cumulus cells and accelerates GVBD
of porcine oocytes. Mol Cell Endocrinol 209, 43-50.
12. Shimada M, Terada T (2001) Phosphatidylinositol 3-kinase in cumulus cells and oocytes is responsible for activation of oocyte mitogen-activated protein kinase during meiotic progression beyond
the meiosis I stage in pigs. Biol Reprod 64, 1106-1114.
13. Petters RM, Reed ML (1991) Addition of taurine or hypotaurine toculture medium improves development of one- and two-cell pig
embryos in vitro. Theriogenology 35, 253 (abstract).
14. Shimada M, Maeda T, Terada T (2001) Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells
are regulated by PKC and PI 3-kinase during meiotic resumption
in porcine oocytes. Biol Reprod 64, 1255-1263.
15. Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT,
Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Garnier J (1989)
Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245, 525-528.
16. Meroni SB, Riera MF, Pellizzari EH, Cigorraga SB (2002) Regulation of rat Sertoli cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway. J Endocrinol 174,
195-204.
17. Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJ(1996) Hormonal regulation of apoptosis in early antral follicles:
follicle-stimulating hormone as a major survival factor. Endocrinology 137, 1447-1456.
18. Wang Y, Chan S, Tsang BK (2002) Involvement of inhibitory nuclear factor-kappaB (NFkappaB)-independent NFkappaB activation in the gonadotropic regulation of X-linked inhibitor of apoptosis expression during ovarian follicular development in vitro.
Endocrinology 143, 2732-2740.

ORIGINAL PAPER

53

Masayuki SHIMADA et al.

19. Racowsky C (1985) Effect of forskolin on maintenance of meioticarrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes. J Reprod
Fertil 74, 9-21.
20. Shimada M, Yamashita Y, Ito J, Okazaki T, Kawahata K, Nishibori
M (2004) Expression of two progesterone receptor isoforms in cu-

54Japanese Journal of Reproductive Endocrinology Vol. 9 2004

mulus cells and their roles during meiotic resumption of porcine
oocytes. J Mol Endocrinol 33, 209-225.
21. Shimada M, Kawano N, Terada T (2002) Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media. Reproduction
124, 557-564.