321ORIGINAL
Effects of activation with a Ca ionophore and roscovitineon the development of human oocytes that failed tofertilize after ICSI
Ayako Suto1, Yuya Yano1, Yuri Yamamoto1, Hiroki Noguchi1, Asuka Takeda1, Shota Yamamoto1, 2, Tomohiro Kagawa1,
Kanako Yoshida1, Kenji Hinokio1, Akira Kuwahara1, Toshiyuki Yasui1, and Takeshi Iwasa1
School of Medicine, Tokushima University, Tokushima, Japan, 2Department of Renal and Genitourinary Surgery, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
1

Abstract : Objective : The effects of oocyte activation with a Ca ionophore and roscovitine (Ca+R), a selective
inhibitor of M-phase promoting factor, on unfertilized oocytes after intracytoplasmic sperm injection (ICSI) or
testicular sperm extraction (TESE)-ICSI were evaluated. Method : Oocytes without pronuclei at 18 hours after
ICSI were judged to be unfertilized and were exposed to the Ca ionophore A23187 (5 μM) with or without roscovitine (50 μM). The activation rate was measured 3, 7, and 18 hours later. Oocytes with two polar bodies andtwo pronuclei with a sperm tail were judged to have been activated. Results : At 18 hours, the activation rates in
the control, Ca ionophore, and Ca+R groups were 3.5% (4 / 112), 26.9% (7 / 26), and 32.1% (17/53), respectively. The
activation rate of the Ca+R group was significantly higher than that of the control and similar to that of the Ca
ionophore group. Among the oocytes that remained unfertilized after TESE-ICSI, the activation rates of the Ca
ionophore and Ca+R groups were 22.2% (2 / 9) and 43.8% (7 / 16), respectively. Conclusions : Sequential treatment
with an Ca ionophore and roscovitine activates oocytes that remain unfertilized after ICSI. In TESE-ICSI, the activation rate tended to be increased by the co-administration of roscovitine with a Ca ionophore. J. Med. Invest.
70 : 321-324, August, 2023
Keywords : Ca ionophore, roscovitine, ICSI, oocyte activation, TESEINTRODUCTION
Ovulated oocytes fall dormant in the middle of meiosis II, but
fertilization causes meiosis to resume and a transition to somatic
cell division. This is called oocyte activation. It is known thatM-phase promoting factor (MPF) is closely involved in meioticarrest and resumption. In ovulated oocytes, MPF activity is
maintained at a high level, which results in meiotic arrest in the
middle of meiosis II. When sperm enters an oocyte, a pulsatileincrease in the calcium (Ca) concentration, called Ca oscillation, is observed ; MPF activity is reduced ; and the oocyte is
activated.
In infertility treatment, intracytoplasmic sperm injection(ICSI) is used in cases of severe male infertility. Recently, however, it has been discovered that there are sperm that are unable
to induce oocyte activation, and even if these sperm are treatedwith ICSI, fertilization cannot be completed. There have been
many reports about the successful fertilization of such oocytes byusing artificial activation methods in combination with ICSI, resulting in live births (1-6). We have tried to develop an artificial
oocyte activation method using a Ca ionophore in combination
with puromycin, a protein synthesis inhibitor (7, 8). This activation method has already been employed in clinical practice by
Murase et al., resulting in live births (5). However, since puromycin is a protein synthesis inhibitor, there is concern that it may
cause malformations or genetic abnormalities when used earlyin development. Thus, in this study we investigated the efficacy
Received for publication October 21, 2022 ; accepted December 10, 2022.
Address correspondence and reprint requests to Yuri Yamamoto,
MD, PhD, Department of Obstetrics and Gynecology, Institute of
Biomedical Sciences, Graduate School, Tokushima University, 3-18-15
Kuramoto-cho, Tokushima 770-8503, Japan and Fax : +81-88-6332630. E-mail : yamamoto.yuri@tokushima-u.ac.jp

of the artificial activation of human oocytes using roscovitine, aselective inhibitor of MPF, instead of puromycin.

MATERIALS AND METHODS

Ovarian Stimulation and ICSI Protocol
Oocytes were retrieved from 29 ICSI cycles in 27 couples at
the Tokushima University Hospital. Twenty of the 27 couples
had male infertility factors, i.e. low sperm concentration and / ormotility, and they were recommended for ICSI. Among them,
6 couples had azoospermic infertility, and testicular sperm
extraction (TESE)-ICSI was performed. Ovarian stimulation
and ICSI were performed according to the method described aspreviously (1, 9). Briefly, ovarian stimulation was carried out
with a gonadotropin-releasing hormone agonist long protocol,
and oocytes were retrieved at 35 hours after the administrationof human chorionic gonadotropin. If at least 2 glowing follicles
were not developed, cycle was canceled. The retrieved oocytes
were cultured for 4 to 5 hours. Then the cells of the cumulus
and corona radiata were removed and the denuded oocytes wererinsed several times. Morphologically intact oocytes were selected for ICSI. Semen was produced by masturbation in most caseand TESE was undertaken in azoospermic couples. Sperm was
prepared and adjusted to appropriate concentration, thereafter
ICSI was carried out. After the ICSI, the oocytes were incubatedfor 16 to 18 hours. Fertilization status was confirmed at 12 to 16
hours and 16 to 18 hours after the ICSI, and oocytes that did notexhibit any pronuclei without a second polar body by 18 hours
were judged to be unfertilized (10). Unfertilized oocytes thatexhibited a first polar body were used for this experiment. Informed consent was obtained from each patient and experimental protocol was approved by the institutional review board of
the University of Tokushima School of Medicine and carried out

The Journal of Medical Investigation Vol. 70 2023

322 A. Suto, et al. Activation with a Ca ionophore and roscovitine is effective in terms of fertilization, especially in the TESE-ICSI treated cases.
in accordance with the guidelines issued by the Japan Society of
Obstetrics and Gynecology (11).

Oocyte Activation
A Ca ionophore (calcium ionophore A23187 mixed calcium
magnesium salt C5149, Sigma-Aldrich, Tokyo) and roscovitine(R7772, Sigma, St. Louis) were dissolved in dimethyl sulfoxide(DMSO ; D4540, Sigma-Aldrich, Tokyo) at concentrations of 1
mg / mL and 10 mg / mL, respectively, and stored at -80°C until
use. At the time of use, the Ca ionophore A23187 was diluted in
m-HTF to a final concentration of 5 μM. This dose of Ca ionophore A23187 was determined by our previous study in which 5
μM of A23187 was enough to activate oocyte with no evidence of
normal fertilization (7). Roscovitine was diluted in human tubal
fluid containing 0.4% bovine serum albumin (HTF ; Cat. No.
9600-001, Nippon Medical & Chemical Instruments Co, Osaka,
Japan) to a final concentration of 50 μM. This dose of roscovitine
was determined by our preliminary examination with serial
dilution experiments.
Unfertilized oocytes were activated by our method, which effectively produced haploid parthenogenones (7, 8). The procedure
was as follows : After ICSI, unfertilized oocytes were transferred to HTF medium containing 4% human serum albumin(HSA ; Sigma) and were preincubated at 37°C in 5% CO2 in air
for 30 minutes. After preincubation, the oocytes were washedthree times with m-HTF and exposed to 100 μL of m-HTF medium containing 5 μM of Ca ionophore A23187 (Sigma ; stored
at 1 mg / mL in dimethyl sulfoxide at 20°C) under paraffin oil(Wako Pure Chemical) on a warm plate (37°C) for 5 minutes.
The oocytes were rinsed several times with fresh HTF medium
and then placed in HTF medium containing 10 mg / mL of roscovitine (Sigma ; stored at 10 mg / mL in DMSO at -20°C) with
4% HSA, before being incubated at 37°C in 5% CO2 in air for
5 hours. Then, the oocytes were washed three times with fresh
HTF medium and cultured in a 100-μL drop of HTF medium
supplemented with 4% HSA at 37°C in 5% CO2 in air underparaffin oil for 13 hours.
After being incubated, the oocytes were examined for the formation of pronuclei and the presence or absence of a second polar
body. Activated oocytes were defined as oocytes that exhibited
two polar bodies and two pronuclei with a sperm tail (2PN2PB)
or oocytes that immediately cleaved, and the activation rate wascalculated as follows : Number of activated oocytes / Number oftreated oocytes × 100. Some of the oocytes that displayed two
pronuclei and a second polar body were additionally incubatedunder the same conditions for 24 hours and then were examined
to determine whether they had cleaved. Cleaved oocytes werescored for quality according to our embryo scoring system (12).

respectively (Table 2). The time-series of these cases demonstrated that the activation rate of the Ca+R group tended to become
higher than that of the Ca ionophore group over time (Figures
1 and 2).
Table 1. The rate of oocyte activation
No. of oocytes No. of activated oocytes
treatedwith 2PN2PB (%)
control112

4 (3.5)

Ca ionophore26

7 (26.9) *Ca ionophore with roscovitine53

17 (32.1) **P < 0.05 vs control

Table 2. The rate of oocyte activation (TESE)
No. of oocytes No. of activated oocytes
treatedwith 2PN2PB (%)
Ca ionophore

92 (22.2)

Ca ionophore with roscovitine16

7 (43.8)

Fig 1. Effects of treatment with a Ca ionophore alone or a Ca
ionophore with roscovitine on the oocyte activation rate at 7 and
16–18 hr after ICSI in the non-TESE-treated cases

Statistics
Statistical analyses were performed using the chi-square test.
Statistical analyses were performed by the software Statcel(v.4 ; OMS Publishing, Inc., Tokorozawa, Japan). P-values of <0.05 were considered statistically significant.

RESULTS
The activation rates of the control, Ca ionophore, and Ca ionophore with roscovitine (Ca+R) groups were 3.5% (4 / 112), 26.9%(7 / 26), and 32.1% (17 / 53), respectively. Significant differences in
the activation rate were observed between the control group andthe Ca ionophore group, and between the control group and the
Ca+R group (Table 1).
In the cases treated with TESE, the activation rates of the Ca
ionophore and Ca+R groups were 22.2% (2 / 9) and 43.8% (7 / 16),

Fig 2. Effects of treatment with a Ca ionophore or a Ca ionophorewith roscovitine on the oocyte activation rate at 7 and 16–18 hr after
ICSI in the TESE-treated cases

The Journal of Medical Investigation Vol. 70 August 2023
DISCUSSION
The present study showed that artificial oocyte activation
with a combination of roscovitine and a Ca ionophore was able
to activate oocytes as well as treatment with the Ca ionophore
alone, even in cases in which TESE was employed, in which Ca
oscillation may be unlikely to occur, and the oocyte activation
rate tended to be higher in the Ca+R group.
It has been reported that fertilization failure occurs in 2-3%
of ICSI cycles and that most cases in which ICSI fails to fertilize
oocytes are due to the failure of oocyte activation (13-15). In addition, it is known that sperm are present in approximately 83%
of oocytes that are determined to be unfertilized after ICSI (16),and if these unfertilized oocytes are properly activated they may
show bipolar anterior nuclei, like normally fertilized oocytes,
after activation and continue to develop normally thereafter. In
fact, there have been many reports of artificial oocyte activation
using chemical agents, such as Ca ionophores or electric pulses,
after ICSI in cases of globozoospermia or repeated fertilization
failure after ICSI, which resulted in pregnancy (1-6). The use of
Ca ionophores or other chemical agents or electric pulses after
ICSI has been reported in many cases of globozoospermia (17).
We have previously developed a method for artificially activating ovulated oocytes in mice, in which we used a combination of a
Ca ionophore and puromycin to obtain a high rate of half-length
eggs (7). In addition, we have reported a method for artificially
activating unfertilized human oocytes using a combination of
a Ca ionophore and puromycin (8). It has been confirmed that
this method activates unfertilized human oocytes after ICSI at
20 hours after the injection of sperm and that the rate of pronucleus appearance was approximately 90%, the division rate wasapproximately 65%, and the percentage of embryos with a goodmorphology was approximately 45% (8, 14). Furthermore, another clinical study showed that oocytes activated with this method
were responsible for live births (5). However, the mechanism bywhich puromycin causes oocyte activation is still unclear, andthere is concern that the use of puromycin, a protein synthesis
inhibitor, early in development may induce malformations or
genetic abnormalities.
On the other hand, since roscovitine is a selective inhibitorof MPF, a complex of cyclin-dependent kinase 1 (Cdk1) andcyclin B, its pharmacological mechanism is evident. Althoughroscovitine exerts its effects by competing for the ATP-binding
domain of Cdk, it does not affect other related kinases, suchas extracellular signal-regulated kinases 1 and ERK2, andmitogen-activated protein kinase, which appears to be involvedin oocyte activation along with MPF (18, 19). Therefore, it isconsidered that the use of roscovitine is safer than the use of puromycin for inducing fertilization and subsequent development.
Although roscovitine inhibits the activity of Cdk2-cyclin A andCdk2-cyclin E, which are involved in the cell cycle (18, 19), theseactions of roscovitine are known to be reversible ; i.e., they cease
just after the removal of roscovitine (18, 20, 21). Furthermore,
although roscovitine inhibits mRNA synthesis by suppressing
Cdk (7, 22) early oocyte development utilizes maternally derived
mRNAs that are present in the oocyte, and hence, the effects ofroscovitine may be small. However, because adverse effects ofroscovitine on oocytes and embryos cannot be completely ruled
out, the dividing ability, developmental potential, etc., of oocytes
subjected to roscovitine treatment should be further evaluatedprior to the drug’s clinical application. Especially, chromosomal
analysis of activated oocyte may be essential to determine thesafety of this method.
In the present study, although there was no significant difference in the oocyte activation rate between the Ca ionophore
alone and Ca+R methods in the cases treated with TESE-ICSI,

323

the activation rate tended to be higher in the cases treated witha combination of roscovitine and a Ca ionophore. This tendency
was observed at 16-18 hours after ICSI, but not at 7 hours after
ICSI. In our preliminary experiment, it was confirmed that the
suppressive action of combined treatment with a Ca ionophoreand roscovitine on MPF activity was sustained longer than wasseen after treatment with the Ca ionophore alone (unpublisheddata). Thus, it is speculated that the lack of reduction in MPF
activity may have caused the low fertilization rate seen in the
TESE-ICSI-treated cases and that longer-lasting effects of roscovitine on MPF may be effective in terms of fertilization. Further
examinations would be needed to confirm this hypothesis.

CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.

REFERENCES
1.

2.

3.

4.

5.

6.

7.

8.

9.

Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter
P, Dhont M : Fertilization and pregnancy after assisted
oocyte activation and intracytoplasmic sperm injection in acase of round-headed sperm associated with deficient oocyte
activation capacity. Fertil Steril 68 : 1144-1147, 1997
Kim ST, Cha YB, Park JM, Gye MC : Successful pregnancy
and delivery from frozen-thawed embryos after intracytoplasmic sperm injection using round-headed spermatozoa
and assisted oocyte activation in a globozoospermic patientwith mosaic Down syndrome. Fertil Steril 75 : 445-447,
2001
Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E,
Gal M, Silber SJ : Successful pregnancy and delivery after
calcium ionophore oocyte activation in a normozoospermic
patient with previous repeated failed fertilization after intracytoplasmic sperm injection. Fertil Steril 79 : 1656-1658,
2003
Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS : Successful
fertilization and pregnancy after intracytoplasmic sperm
injection and oocyte activation with calcium ionophore in anormozoospermic patient with extremely low fertilization
rates in intracytoplasmic sperm injection cycles. Fertil Steril 82 : 475-477, 2004
Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M,
Yoshizawa M, Araki Y : Pregnancy following chemical
activation of oocytes in a couple with repeated failure of
fertilization using ICSI : case report. Hum Reprod 19 : 16041607, 2004
Heindryckx B, Van der Elst J, De Sutter P, Dhont M : Treatment option for sperm- or oocyte-related fertilization
failure : assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod 20 : 2237-2241, 2005
Nakasaka H, Yamano S, Hinokio K, Nakagawa K, Yoshizawa
M, Aono T : Effective activation method with A23187 andpuromycin to produce haploid parthenogenones from freshly
ovulated mouse oocytes. Zygote 8 : 203-208, 2000
Nakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T : Effect of activation with Ca ionophore
A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm
injection. Fertil Steril 76 : 148-152, 2001
Van Steirteghem AC, Nagy Z, Joris H, Liu J,Staessen C,
Smitz J, Wisanto A, Devroey P : High fertilization andimplantation rates after intracytoplasmic sperm injection.
Hum Reprod 8 : 1061-1066, 1993

324 A. Suto, et al. Activation with a Ca ionophore and roscovitine is effective in terms of fertilization, especially in the TESE-ICSI treated cases.
10. Nagy ZP, Liu J, Joris H, Van Steirteghem AC : Time-courseof oocyte activation, pronucleus formation and cleavage inhuman oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod 9 : 1743-1748, 1993
11. Guideline on the experimental use of human sperm, oocytes
and embryos. Acta Obstet Gynaecol Jpn 3 : 85, 1985
12. Nakagawa K, Yamano S, Kamada M, Hinokio K, Maegawa
M, Aono T : Quality of embryo does not affect the implantation rate of IVF-ET in infertile women with antisperm
antibody. Fertil Steril 72 : 1055-1060, 1999
13. Sousa M, Tesarik J : Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum
Reprod 9 : 2374-2380, 1994
14. Tesarik J, Sousa M : More than 90% fertilization rates after
intracytoplasmic sperm injection and artificial inductionof oocyte activation with calcium ionophore. Fertil Steril
63 : 343-349, 1995
15. Mahutte NG, Arici A : Failed fertilization : is it predictable?
Curr Opin Obstet Gynecol 15 : 211-218, 2003
16. Flaherty SP, Payne D, Matthews CD : Fertilization failures
and abnormal fertilization after intracytoplasmic sperm
injection. Hum Reprod 13 : 155-164, 1998
17. Lu Q, Zhao Y, Gao X, Li Y, Ma S, Mullen S, Critser JK,

Chen ZJ : Combination of calcium ionophore A23187 withpuromycin salvages human unfertilized oocytes after ICSI.
Eur J Obstet Gynecol Reprod Biol 126 : 72-76, 2006
18. Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki
N, Inagaki M, Delcros JG, Moulinoux JP : Biochemical andcellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.
Eur J Biochem 243 : 527-536, 1997
19. De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M,
Kim SH : Inhibition of cyclin-dependent kinases by purine
analogues : crystal structure of human cdk2 complexedwith roscovitine. Eur J Biochem 243 : 518-526, 1997
20. Hajdúch M, Havlíèek L, Veselý J, Novotný R, Mihál V,
Strnad M : Synthetic cyclin dependent kinase inhibitors.
New generation of potent anti-cancer drugs. Adv Exp Med
Biol 457 : 341-353, 1999
21. Sielecki TM, Boylan JF, Benfield PA, Trainor GL : Cyclin-dependent kinase inhibitors : useful targets in cell cycle
regulation. J Med Chem 43 : 1-18, 2000
22. Ljungman M, Paulsen MT : The cyclin-dependent kinase
inhibitor roscovitine inhibits RNA synthesis and triggers
nuclear accumulation of p53 that is unmodified at Ser15 andLys382. Mol Pharmacol 60 : 785-789, 2001