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Received: 7 July 2017 Accepted: 26 September 2017 DOI: 10.1111/rda.13105
ORIGINAL ARTICLE
The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage ECS Santos1 | R Appeltant2 | TQ Dang-Nguyen2 | J Noguchi2 | H Kaneko2 | K Kikuchi2 | T Somfai1 1 Institute of Livestock and Glassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan 2
Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan Correspondence Tamás Somfai, Institute of Livestock and Glassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan. Email:
[email protected] Present addresses: Ruth Appeltant, Unit of Animal Genomics, GIGA-R, University of Liège (B34) Liège, Belgium Funding information CNPq-Brasil, Grant/Award Number: 205473/2014-8; Japan Society for the Promotion of Science; KAKENHI, Grant/ Award Number: 26870839
Contents We tested the effects of resveratrol both as a pre-treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre-treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non-vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non-vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification-related damages.
1 | INTRODUCTION
fertilization (oocyte activation). Nevertheless, the exact nature of these sublethal damages is still unclear. Recent investigations showed
Oocyte cryopreservation is a desired approach for the maintenance of
that cytoplasmic maturation and energy balance of immature porcine
female germplasm in ex situ gene banks for farm animals (Woelders,
oocytes were not affected by vitrification (Appeltant et al., 2017).
Windig, & Hiemstra, 2012). In pigs, although the technologies for the
However, it has been demonstrated previously that vitrification trig-
in vitro maturation (IVM) and fertilization (IVF) of oocytes and in vitro
gers the apoptotic cascade in matured (MII stage) porcine oocytes
production of embryos (IVP) have progressed (Grupen, 2014) and live
(Vallorani et al., 2012). Apoptotic events can be triggered in oocytes
offspring have been produced from porcine oocytes vitrified at the
by various pathways leading to DNA damage and eventually cell death
immature stage (Somfai et al., 2014), still only a small percentage of
(Tiwari et al., 2015). Resveratrol (3,4,5-trihydroxy-trans-stilbene), a
vitrified oocytes can develop to the blastocyst stage. Relatively high
phytoalexin with anti-apoptotic and antioxidant properties, was re-
survival and maturation rates (both above 70%) of vitrified immature
ported to modulate the apoptotic process and reduced the frequency
oocytes suggest that sublethal damages (i.e., damages which do not
of apoptosis in porcine oocytes vitrified at the MII stage (Giaretta,
lead to imminent cell death) caused by vitrification hamper develop-
Spinaci, Bucci, Tamanini, & Galeati, 2013). On the other hand, there are
mental competence which is manifested only after maturation and
fundamental differences in the sensitivity to cryopreservation and the
Reprod Dom Anim. 2017;1–9.
wileyonlinelibrary.com/journal/rda © 2017 Blackwell Verlag GmbH | 1
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SANTOS et al.
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response to the vitrification procedure between mature and immature
polyvinylpyrrolidone (P-0930, Sigma-Aldrich), 0.3 M sucrose (196-
porcine oocytes (Somfai, Kikuchi, & Nagai, 2012). The aim of this study
00015, Wako Pure Chemical Industries, Osaka, Japan), 17.5% (v/v)
was to investigate whether resveratrol can improve the developmental
EG and 17.5% (v/v) PG. Treatment of COCs in VS medium (includ-
competence of porcine oocytes vitrified at the immature germinal ves-
ing washing, loading and removal of excess VS) did not exceed 40
icle stage. We tested the effects of resveratrol both as a pre-treatment
s. Vitrified samples were stored in a LN tank until use. Warming of
before cryopreservation and as a recovery treatment after warming
vitrified COCs was performed according to a previous report (Somfai
during IVM on the viability, maturation, developmental competence,
et al., 2015) with slight modification. In brief, Cryotop sheets were im-
cytoplasmic redox status and apoptosis of porcine oocytes vitrified at
mersed directly into 2.5 ml of warming solution (0.4 M Sucrose in BM)
the immature stage.
in a 35-mm plastic dish (Falcon 351008, Becton Dickinson, Franklin Lakes, NJ, USA) for 1 min at 42°C. The COCs were then consecutively
2 | MATERIALS AND METHODS 2.1 | Ethics statement
transferred for periods of 1 min (each) to 500-μl droplets of BM supplemented with 0.2, 0.1 and 0.05 M of sucrose at 38.0°C. The warmed COCs were washed in BM without sucrose at 38.0°C and then placed into maturation medium.
Live animals raising ethical concerns were not used in this study.
2.2 | Collection of cumulus–oocyte complexes (COCs)
2.4 | In vitro maturation (IVM) The maturation culture medium was porcine oocyte medium (POM, Yoshioka, Suzuki, & Onishi, 2008) supplemented with 10 IU/ml eCG
Porcine ovaries of cross-bred gilts (Landrace × Large White) were
(Serotropin; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan), 10 IU/ml
randomly selected from a local slaughterhouse, stored at 35–37°C
hCG (Puberogen, Novartis Animal Health, Tokyo, Japan) and 10 μg/ml
after washing with Dulbecco’s phosphate buffered saline (PBS) and
epidermal growth factor (EGF, Sigma-Aldrich). After warming, all
transported to the laboratory within 1–2 hr. Upon arrival, COCs
COCs were washed three times and cultured in 500 μl of aliquots of
were collected by scraping of 2- to 6-mm follicles with a fine surgi-
maturation medium supplemented with 1 mM dibutyryl cAMP (db-
cal blade in a collection medium, which was medium 199 (M199 with
cAMP; Sigma-Aldrich) in 4-well dishes (Nunc, Nunclon Delta Surface,
Hanks’ salts; Sigma-Aldrich, St. Louis, MO, USA) supplemented with
Thermo Fisher Scientific, Roskilde, Denmark) without oil coverage,
5% (v/v) of foetal bovine serum (FBS, Gibco, Thermo Fisher Scientific,
each well containing 40–50 COCs for 22 hr in 5% CO2, 5% O2 and
Life Technologies, Carlsbad, CA, USA), 20 mM of HEPES (Dojindo
90% N2 at 39°C. Subsequently, they were cultured for an additional
Laboratories, Kumamoto, Japan) and antibiotics [100 IU/ml of strep-
22–24 hr in POM medium containing hormones and EGF but with-
tomycin sulphate (Sigma-Aldrich), 100 IU/ml penicillin G potassium
out dbcAMP under the same conditions as during the first 22 hr of
(Sigma-Aldrich)]. After dissection, COCs with fully grown oocytes with
IVM.
at least two layers of cumulus cells were selected for the subsequent experiments.
2.3 | Vitrification and warming of immature COCs Cryoprotectant treatment regimen before the vitrification of COCs
2.5 | Evaluation of survival and maturation of oocytes After IVM, COCs were treated with a solution of 150 IU/ml hyaluronidase (Sigma-Aldrich) in collection medium for 30 s and then trans-
was performed according to a previous report (Somfai et al., 2015).
ferred to collection medium in which cumulus cells were removed
In brief, a group of 50–70 COCs were incubated in 1 ml of a basic
from the oocytes by gentle pipetting with a fine glass pipette. Then
medium (BM) for 30 min, which was a modified glucose-free North
the live/dead status and maturation of the oocytes were assessed
Carolina State University (NCSU)-37 medium (Petters & Wells,
morphologically under a stereo microscope. Oocytes with a normal
1993) supplemented with 20 mM HEPES, 0.17 mM sodium pyru-
spherical shape and dark granulated cytoplasm surrounded by a mem-
vate, 2.73 mM sodium lactate, 50 μM ß-mercaptoethanol and 4 mg/
brane with a definite smooth surface were considered alive. Oocytes
ml bovine serum albumin (Fraction V, Sigma-Aldrich). The BM me-
without a definite membrane surface and/or with brownish cyto-
dium was further supplemented with 7.5 μg/ml cytochalasin B (CB;
plasm were considered dead. Live oocytes with the first polar body
C-6762, Sigma-Aldrich). Then the COCs were transferred into an
(1PB) extruded were considered matured and used in subsequent
equilibration solution (ES) comprised of BM supplemented with
experiments.
7.5 μg/ml CB, 2% (v/v) ethylene glycol (EG) and 2% (v/v) propylene glycol (PG) for 5–15 min at 38.5°C. Afterwards, 10–12 COCs were washed two times in 50 μl of vitrification solution (VS) at 38.0°C,
2.6 | Parthenogenetic activation (PA) of oocytes
loaded on Cryotop sheets (Kitazato, Biopharma, Shizuoka, Japan) in
The matured oocytes were placed between a pair of electrodes of an
a minimum volume (
