9HWHULQDU\ 6FLHQFH on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle Min Kyu Kim 1 , Yuda Heru Fibrianto 1 , Hyun Ju Oh 1 , Goo Jang
Trang 19HWHULQDU\ 6FLHQFH
on in vitro maturation of canine oocytes collected from dogs with different
stages of the estrus cycle
Min Kyu Kim 1
, Yuda Heru Fibrianto 1
, Hyun Ju Oh 1
, Goo Jang 1
, Hye Jin Kim 1
, Kyu Seung Lee 2
, Sung Keun Kang 1,3
, Byeong Chun Lee 1,3,
*, Woo Suk Hwang 1,3,4
1Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
2
Department of Animal Science, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea
3
The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, Korea
4
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
maturation (IVM) medium was shown to improve embryo
development and quality in several species Epidermal
growth factor (EGF) was also shown to improve IVM of
human oocyte and embryo development after in vitro
fertilization (IVF) The effect of these two compounds were
suggested to be mediated through the synthesis of
glutathione (GSH) which is known to play an important role
in protecting the cell or embryos from oxidative damage.
Thus, it is suggested that supplementation of canine IVM
positive role in IVM of various mammalian oocytes and
embryo development, including cattle, pigs, rodents and
humans This study investigates the effect of ovarian estrus
stage on canine oocyte quality and supplementation of
results, a significantly higher percentage of oocytes
β-ME supplemented oocytes collected from the follicular stage.
The maturation rate to metaphase I (MI) stage was also
significantly higher in oocytes collected from follicular stage
experimental groups After IVM culture, oocytes recovered
from dogs with the follicular stage and matured in TCM-199
supplemented with 20 ng/ml EGF yielded better oocyte
maturation to MII phase compared to other groups Taken
(20 ng/ml) improved IVM of canine oocytes to MII stage.
factor (EGF), canine oocytes, in vitro maturation (IVM).
Introduction
The efficiency of in vitro maturation (IVM) of canine
oocytes is still very low compared to that found in other mammalian species Small proportions of canine oocytes completed nuclear maturation after being cultured for 48 to
72 h in vitro [20,27,30] Low maturation rates could be due
to either suboptimal culture conditions or low meiotic competence of the oocytes [7] Studies are in progress to determine both the culture requirements for canine oocytes and some parameters indicative of maturation competence The morphological appearance of cumulus-oocyte complexes (COCs) and oocyte diameter have been identified as parameters indicative of maturation competence [14] In contrast to most mammals, bitches ovulate immature oocytes that require 2-5 days for the completion of meiosis within the oviduct [16] and remain surrounded by a tight and multilayered cumulus cell mass [28] The immature stage of oocytes at ovulation and the persistence of cumulus cells during transport and maturation within the oviduct, indicating that investigation of the relationship between cumulus cells and oocytes should help to clarify the reasons for the low IVM rates of canine oocytes This results also suggests the importance of selecting good quality of COCs
by morphological appearance for IVM of oocytes
Increased oxidative stress is one of the causes of impaired IVM of oocyte The GSH is the major nonprotein sulphynyl compound in mammalian cells and is known to play an important role in protecting the cell from oxidative damage
thiol and supplementation of β-ME in IVM medium has
shown to increase intracellular glutathione (GSH) content in bovine oocytes and to improve embryo development and quality in several species [24,25,29] Moreover, the presence
of thiol compound in a maturation medium increased the GSH level and improved developmental competence of pig
*Corresponding author
Tel: +82-2-880-1269; Fax: +82-2-884-1902
E-mail: bclee@snu.ac.kr
Trang 2chromatin decondensation and male pronucleus formation
after sperm penetration, supporting its possible role in
encouraging oocyte cytoplasmic maturation [8] A mechanism
by which β-ME promotes intracellular GSH synthesis was
proposed by Takahasi et al [32] In lymphocytes, β-ME
promotes the uptake of cysteine, enhancing GSH synthesis
[18] It has been suggested that GSH protects cells from
oxidative damage [9,26,34] Thus, increased intracellular
GSH induced by β-ME may provide embryos with a better
intracellular condition, preventing oxidative damaged
possibly promoting late embryonic development
Several hypotheses suggest that epidermal growth factor
(EGF) acts on cells as both a paracrine and autocrine
regulator of ovarian function [3,12] The EGF is one of
many growth factors found throughout the body, including
growing antral follicles within the ovary, and has shown to
stimulate DNA synthesis and cell proliferation in porcine
granulosa cells The EGF also improves IVM of human
oocyte and embryo development after in vitro fertilization
(IVF) [11] Supplementation of canine IVM medium with
EGF may be of benefit due to its positive role in various
other mammalian IVM and embryo culture systems
including cattle, pigs, rodents and humans [6,15,22] In the
serum of both pigs and humans, EGF levels have been
reported at 8 ng/ml and 1.2-3.75 ng/ml, respectively, while
in the follicular fluid, EGF level were varied from 2 to 15
ng/ml in both species [22] The EGF was reported to cause
an increase in the number of mouse oocytes undergoing
germinal vesicle breakdown (GVBD), and to cause cumulus
expansion in bovine and mouse COCs
Consequently, this study investigates the effect of ovarian
estrus stage on oocyte quality and supplementation of
medium with β-ME or EGF on IVM of canine oocytes
Materials and Methods
Oocyte collection
Reproductive tracts from normal bitches greater than 6
months of age were collected after routine ovariohysterectomy
at private clinics, placed immediately into physiological
saline solution (PSS) at 37o
C and transported back to the laboratory within 1 h Ovaries were removed from the tract
and washed free from blood in fresh PSS, and then
repeatedly slashed with a #10 scalpel blade or shaving blade
medium consisting of M-199 with 25 mM HEPES (Sigma,
St Louis, MO) supplemented with 1% fetal calf serum (FBS, Life technology, Rockville, MD) and 1% penicillin-streptomycin (P/S) comprised of 10,000 U/ml of penicillin
G sodium and 10,000µg/ml streptomycin sulfate (Sigma)
Only grade I & II oocytes >100µm in diameter were
selected and used for the experiments Oocytes were washed
3 times in the bench medium to wash off blood and other debris prior to transfer to maturation medium
Removal of cumulus cell and assessment of meiotic stage
At the end of the maturation culture, oocytes were completely denuded via gently pipetting with a fine bore glass pipette and a solution of 0.2% (w/v) hyaluronidase (Sigma) in the bench medium The oocytes were then fixed
in a 3.7% formaldehyde-Triton X-100 (Sigma) solution for
10 min, washed in PBS for an additional 3 min, mounted on
a slide and stained with 1.9µM Hoechst 33342 (Sigma) in
glycerol (Sigma) Oocytes were then evaluated under UV light to determine the stage of meiosis as defined in Table 1 and Fig 1
Experimental design
Supplementation of β-ME in TCM-199 (Experiment 1).
In this experiment, whether β-ME promotes completion
of nuclear maturation in canine oocytes was evaluated The concentrations of β-ME tested were 0.0 (control), 25, 50 and
evaluated for nuclear meiotic stage as previously defined in Table 1 and Fig 1
EGF supplementation in TCM-199 (Experiment 2).
In this experiment, the effect of supplementation of EGF
to basic maturation medium on IVM of canine oocytes was evaluated as previously defined in Table 1 and Fig 1 The concentrations of EGF tested were 0.0 (control), 10 and 20 ng/ml
Statistical analysis
Multiple comparisons (LSD) were implemented using Generalized Linear Models in the SAS 8.12 program When the significant effect in each experimental parameter was detected, subsequent comparison was made by the least
Table 1 Definition of meiotic classification in canine oocytes stained after in vitro maturation
Germinal vesicle (GV) Germinal vesicle (nuclear envelope) still intact; scattered chromatin
Germinal vesicle break down (GVBD)Organization and condensation of chromatin into chromosomes
Metaphase I (MI) Alignment of chromosomes on meiotic spindle
Metaphase II (MII) Meiotic division resulting in chromosome number reduction and expulsion of first polar body Degenerate/dead Abnormal chromosomal configuration/no visible chromatin material
Trang 3square method Difference among the treatment were
considered statistically significant when the p-values were
less than 0.05
Result
The result of Experiment 1 showed that a significantly
higher percentage of oocytes progressed to metaphase II
(MII) (20 or 13%, respectively) in 50 or 100µM β-ME
supplemented oocytes collected from the follicular stage
(Table 2) The maturation rate to metaphase I (MI) stage was
also significantly higher in oocytes collected from follicular
respectively) compared to that observed in other experimental
groups (p < 0.05).
EGF supplementation in TCM-199
In Experimental 2, after 72 h culture, oocytes recovered
from the follicular stage and matured in TCM-199
supplemented with EGF 20 ng/ml yielded better oocyte
maturation to MII phase compared to other groups (13 % vs
3 to 6 %, respectively) (Table 3)
Discussion
Unlike other mammalian oocytes, ovulated canine oocytes complete meiotic maturation within 2-5 days in the oviduct after ovulation [4,16] At present, IVM of canine oocytes is characterized by low and greatly variable success rates [7] Studies have been performed to improve the rate of IVM in canine oocytes by supplementing the culture medium with various serum [25,29], gonadotrophin [15] or steroid hormone [15] The present study investigated the effect of
β-ME or EGF supplementation on the base of the stages of the estrus cycle of the ovaries, and demonstrated that β-ME or
EGF supplementation significantly increased maturation of canine oocyte to MII stage
A number of studies have demonstrated an effect of stage
of the estrous cycle on the meiotic competence of canine
oocytes matured in vitro Yamada et al [35] reported that
32% of preovulatory oocytes collected from superovulated bitches reached MII after 72 h of culture, whereas oocytes from anestrus bitches showed no tendency to resume meiosis even after a culture period of up to 144 h In a
similar investigation by Luvoni et al [23], a high percentage
of the oocytes collected during anestrus were in the germinal
Fig 1 Canine oocytes stained with Hoechst 33342 and visualized under UV light Germinal vesicle (A), germinal vesicle breakdown
(B), metaphase I (C) and metaphase II (D) (x200)
Trang 4vesicle breakdown (GVBD) stage at the time of collection.
Such oocytes could be derived from atretic follicles, which
are usually ruptured by the slicing procedure used for
collection It is generally accepted that GVBD can occur
spontaneously in human atretic follicles [11] In contrast,
Hewitt and England [15] reported that no significant
differences in maturation rates were observed between
oocytes collected at proestrus and estrus stages In the
present study, higher proportion of oocytes collected from
the follicular stage ovaries reached MI and MII stage in the
absence or presence of β-ME or EGF compared to those
collected from ovaries of anestrus and luteal stages Our
results indicate the importance of oocytes recovery phase to
select potentially meiotically competent canine oocytes for
use of in vitro experiment.
Glutathione (GSH) is the major non-protein sulfydryl
compound present in mammalian cells Multiple actions
have been described for GSH, including increasing amino acid transport, stimulating DNA and protein synthesis, reduction of disulfides and protection against toxic effects of
oxidative damage [21] Takahashi et al [33] demonstrated
that the supplementation of low molecular weight thiol compound, to culture medium can increase the
cystein-mediated GSH synthesis in bovine embryo produced in vitro Similarly, addition of β-ME increased intracellular
GSH in mouse lymphocytes [37] It has been shown that this thiol-compound stimulates glutathione synthesis [24], which, in turn, protects the bovine oocytes from oxidative damage Addition of β-ME to chemical defined maturation
medium also increased the percentage of ovine oocytes completing nuclear maturation [25] The result of the present study demonstrated that supplementation of canine IVM medium with β-ME at 50 or 100 µM had beneficial
effects on meiotic competence of oocytes collected from the
Estrus stages β-ME (µM) No of oocytes examined % nuclear status of oocytes (mean± SD)
Anestrus
39.2± 6.8 00.0± 4.7a
00.0± 2.4a
37.0± 9.3 14.8± 6.5ab
00.0± 3.3a
40.0± 6.0 13.8± 4.2b
03.0± 2.1a
52.1± 7.1 6.5± 5.0a
00.0± 2.5a Follicular
44.4± 8.1 16.6± 5.6bc
00.0± 2.8a
52.3± 10.6 28.5± 7.4c
00.0± 3.7a
46.6± 8.9 16.6± 6.1bc
20.0± 3.1b
25.8± 8.7 32.2± 6.0c
12.9± 3.1b
Luteal
36.9± 5.3 13.0± 3.7b
02.3± 1.8a
37.6± 5.8 17.3± 4.0bc
02.8± 2.0a
29.7± 5.3 11.9± 3.7b
03.5± 1.8a
43.0± 6.0 10.7± 4.2a
01.5± 2.1a a-c
Values with different superscripts are significantly different (p < 0.05)
Table 3 Meiotic status of canine oocytes recovered from different ovarian estrus stages and cultured in TCM-199 supplemented with
various concentrations of epidermal growth factor (EGF)
Estrus stages EGF (ng/ml) No of oocytes
examined
% nuclear status of oocytes (mean± SD)
Anestrus
47.5± 5.5 08.7± 3.8a
00.0± 1.9a
46.8± 4.4 07.9± 3.0a
00.0± 1.5a
39.3± 4.4 15.5± 3.1ab
01.6± 1.5a Follicular
44.4± 8.2 11.1± 5.7ab
02.7± 2.8ac
29.4± 8.5 23.5± 5.8b
05.8± 2.9abc
33.3± 7.3 20.0± 5.1b
13.3± 2.5b
Luteal
54.1± 10.1 12.5± 7.0ab
04.1± 3.5ac
51.6± 8.9 12.9± 6.1ab
03.2± 3.1ac
47.2± 8.2 27.7± 5.7b
08.3± 2.8bc a-c
Values with different superscripts are significantly different (p < 0.05)
Trang 5follicular stage, consistent with the result from Takahashi et
al [32] in bovine IVM As described above, it is important
to mention that the outcome of our experiments was most
likely influenced by the stage of the estrus cycle of the dogs
from which the oocytes were collected; the positive effect of
β-ME on IVM of canine oocytes was only observed in the
oocytes collected from the follicular stage
It is well established that the addition of EGF promote in
vitro oocyte maturation, and also improve fertilization and
cleavage rates in many species, including the pig, rat, mice
[5], fox [31], cow [19], and humans [17] In addition to
accelerating nuclear maturation in the oocyte, literature also
suggests that the addition of EGF improves cytoplasmic
maturation [20] Furthermore, studies indicate that EGF
induces oscillations in calcium efflux in COCs of mice, and
also promotes the synthesis of GSH within the oocyte [8]
The role of calcium oscillations in immature oocytes is not
as widely studied as it is in fertilization events, but it is
suggested to have a role in resumption of meiosis, as
described in research on amphibian and invertebrate oocytes
[15] The results of the experiments presented in this study
demonstrated the benefit of supplementing canine IVM
medium with EGF Supplementation of 20 ng/ml EGF in
TCM-199 showed significantly improved rate of meiotic
competence of oocytes collected from follicular stage In
support of our results, a study reports a significant increase
in the blastocyst development from oocytes that were
cultured in serum-free medium supplemented with 10 ng/ml
EGF, and this was attributed to an increase in GSH synthesis
influenced by EGF [2] It is also important to mention that
the outcome of our experiments was most likely influenced
by the stage of the estrus cycle of the dogs; the positive
effect of EGF supplementation on IVM of canine oocytes
was only observed in the oocytes collected from the
follicular stage
In conclusion, supplementation of canine IVM medium
with GSH-synthesis stimulator, β-ME or EGF improved
IVM of canine oocytes collected from dogs with the
follicular stage of the estrus cycle
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