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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 3, Num. 1, 2005, pp. 36-41
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EFFECTS OF AMINO ACIDS DURING BOVINE OOCYTE MATURATION ON SUBSEQUENT
EMBRYONIC DEVELOPMENT IN VITO
Iranian Journal of Reproductive Medicine Vol.3. No.1, 2005,
pp: 36-41
Effects of essential and non-essential amino acids on in-vitro maturation, fertilization and development of immature bovine oocytes
Nourollah Rezaei,¹ Ph.D., Ri-Cheng Chian,² Ph.D.
1 Assistant professor, Department of Anatomy &
Embryology, Mazandaran University of Medical Sciences, Sari, Iran. 2 Assistant professor, Division of Reproduction
Biology, Department of Obstetrics & Gynecology, Mcgill University,
Montreal, Quebec, Canada.
Corresponding Author: Dr Nourollah Rezaei, Department of Anatomy &
Embryology, Sari Medical Faculty, Mazandaran University of Medical Sciences, Khazar
Boulevard, Sari, Mazandaran, Iran. Email: noor454@yahoo.com.
Code Number: rm05007
Abstract
Background:
Addition of amino acids to the culture medium is beneficial for embryonic
development in many species.
Objective:
The objective of this study was to investigate the effects of amino acids
on the in vitro maturation and embryonic development of the bovine oocyte.
Materials
and Methods: Bovine ovaries were collected from a local abattoir and
brought into laboratory. Cumulus-oocyte complexes (COCs; n=1212) were aspirated
from follicles (2-8 mm in diameter) and randomly assigned to four groups for
maturation in culture: (1) Basic medium alone as control; (2) Basic medium
supplemented with 2% MEM essential amino acids solution; (3) Basic medium
supplemented with 1% MEM non-essential amino acids solution; and (4) Basic
medium supplemented with 2% MEM essential amino acids solution + 1% MEM
non-essential amino acids solution. COCs were incubated in 1 ml maturation
medium in an Organ culture dish at 38.5°C in an atmosphere of 5% CO2 with high
humidity. After 24 h of culture, 372 oocytes were fixed to determine maturation
rate and the remaining oocytes were used for in vitro fertilization (IVF).
Following 18 h of insemination, 437 oocytes were fixed and examined for
fertilization and 403 oocytes were further cultured.
Results:There were no differences in maturation rates and
penetration rates among the four groups. Although oocyte cleavage rates were
not different in the four groups, embryo development up to the 8-cell stage and
blastocyst were significantly higher (p<0.05) in Group (2) and (4) than in
the Control and Group (3).
Conclusion:
These results indicate that the presence of amino acids, especially essential
amino acids in the maturation medium is beneficial to oocyte cytoplasmic
maturation and subsequent early embryo development in vitro.
Key words: In vitro maturation, Bovine oocyte, Amino acids.
Introduction
Essential and/or non-essential amino acids are
commonly added to serum-supplemented or serum-free culture media used for
mammalian embryo development in vitro. In many species, it has been known that
addition of amino acids to the culture medium is beneficial for embryonic
development (1-3). Apart from amino acids used for protein synthesis, they play
important role as osmolytes (4), intracellular buffers (5), heavy metal
chelators and energy sources as well as precursors for versatile physiological
regulators, such as nitric oxide and polyamines (6). It has also been shown
that the culture medium with amino acids affect glucose metabolism in mouse
blastocysts in vitro (2). Although it has been shown that amino acids support
rabbit (7), hamster (8), porcine (9) and bovine (10) oocyte maturation, amino
acid requirements for oocyte maturation in culture is not fully understood. The
objective of this study was to investigate the effects of essential and
non-essential amino acids on in-vitro maturation, subsequent fertilization and
embryo development of immature bovine oocyte.
Materials and
Methods
Maturation of Oocytes in Vitro
This study was an experimental type. Ovaries from
Holstein heifers and cows were collected at local abattoir shortly after
slaughter and transported to the laboratory within 3 h in 0.9% NaCl aqueous
solution containing 100 IU/ml penicillin, and 100µg/ml streptomycin at
approximately 35°C. Cumulus-oocyte complexes (COCs; n=1212) were aspirated from
2 to 8 mm follicles with an 18 G needle connected to a 10ml disposable syringe.
Oocytes with unexpanded cumulus mass, having more than two layers of cumulus
cells and with homogeneous granular ooplasm were selected, as described
previously (11). The COCs were rapidly washed 4 times in HEPES buffered
Tyrodes medium (TLH) supplemented with 0.3% polyvinylpyrrolidone (PVP), 0.25mM
pyruvic acid (Sigma) and 50 µg/ml gentamycin (Sigma). The basic medium for
oocyte maturation is a chemically defined protein-free medium (Table I) supplemented with 0.3% PVP (Sigma), 75mIU/ml
FSH and LH (Humegon; Organon, Scarborough, ON, Canada).
Table I. Composition of basic medium for oocyte maturation
Component
|
mg/L
|
NaCl
|
6800.00
|
KCl
|
400.00
|
NaH2PO4·H2O
|
125.00
|
NaHCO3
|
1250.00
|
CaCl2
|
200.00
|
MgSO4
|
98.00
|
D-Glucose
|
1000.00
|
L-Glutamine
|
292.00
|
Sodium Pyruvate
|
110.00
|
Phenol Red
|
5.00
|
Penicillin
|
50.00 IU
|
Streptomycin
|
50.00 µg
|
After washing, COCs were randomly assigned to
following 4 groups of maturation medium respectively: (1) Basic medium alone
(Control); (2) Basic medium supplemented with 2% MEM essential amino acids
solution (GIBCO; 50X); (3) Basic medium supplemented with 1% MEM non-essential
amino acids solution (GIBCO; 100X); (4) Basic medium supplemented with 2% MEM
essential amino acids solution + 1% MEM non-essential amino acids solution.
COCs were cultured in 1 ml maturation medium in an Organ culture dish (Falcon;
60x15 mm) at 38.5°C in an atmosphere of 5% CO2 with high humidity.
After maturation for 24 h, some oocytes (n=372) were fixed and stained, then
evaluated for stage of nuclear maturation by bright field microscopy
(magnification: 400X) to examine maturation rate. The stages of nuclear
maturation were assessed as germinal vesicle (GV: oocytes arrested at prophase
I of meiosis are characterized at the light microscope level, as having a
visible nucleus), metaphase I (MI: when meiosis resumes, the oocyte undergoes
dissolution of the nuclear envelope, subsequently chromatin condenses into
discrete bivalents that align on the meiotic spindle at metaphase I) and
metaphase II (MII: the separation is complete at MII, which is recognizable at
the light microscope level by the presence of the first polar body) (11). The remaining
oocytes (n=840) were used for in vitro fertilization (IVF).
Sperm Preparation and IVF
Frozen semen was used for IVF. Straws of semen were
thawed in a water bath (35°C) for 30 seconds and processed by swim-up as
described before (11). The sperm were then washed twice in modified Tyrodes
albumin lactate pyruvate medium (Sp-TALP) used for sperm culture containing 6
mg/ml fatty acid-free BSA (Sigma), 10mM pyruvic acid, and 50µg/ml gentamycin.
Following maturation, COCs were washed three times with TLH and then sperm/oocytes
were incubated in 50µl droplets of the fertilization medium, modified Tyrodes
medium (mTALP), and contained 2µg/ml heparin under mineral oil at 38.5°C in 5%
CO2 with high humidity. The final sperm concentration of 1 x 106
sperm/ml was used for oocyte insemination and five oocytes were used for each
50µl droplet.
Embryo developmental culture
Following 18 h of insemination, some oocytes
(n=437) were fixed for examining fertilization rate and the remaining oocytes
(n=403) were washed three times with TLH and then transferred to 50µl droplets
of development medium (BECM: Bovine Embryo Culture Medium) supplemented with 3
mg/ml BSA (fatty acid-free, Sigma) and 1µg/ml gentamycin under mineral oil. The
culture medium was changed at 24 h intervals until 120 h after insemination and
then the embryos were transferred to 50µl droplets of BECM supplemented with
10% FBS and 0.25mM pyruvic acid for further developmental culture (8days).
Fixation of oocytes
At 24 h of maturation and at 18 h of
insemination, the oocytes were mounted on slides with coverslips and fixed with
acetic acid/ethanol (1:3) solution for at least 24 h. The oocytes were then
stained with 1% orcein dissolved in 45% acetic acid solution and examined for
evidence of fertilization. Fertilization was identified by observing two
pronuclei with an accompanying sperm tail in the cytoplasm. Oocytes with two
pronuclei and a clear second polar body but without a sperm tail were also
considered to have been fertilized. Oocytes with a female pronucleus and a
decondensed sperm head were considered abnormal fertilization. Oocytes with
three pronuclei (two sperm) or more were considered polyspermy.
Statistical analysis
The numbers of immature oocytes, maturation,
fertilization and embryo cleavage rates as well as blastocyst formation rate from
each group were analyzed by one-way analysis of variance. When analysis
revealed significance, the groups were compared using the Student-Newman-Keuls
test.
Results
As shown in Table II when the immature bovine
oocytes were cultured in basic IVM- medium supplement with 2% essential and 1%
non-essential amino acids, there were no significant differences in maturation
rates among the four groups.
Table III shows the effect of IVM-medium
supplement with 2% essential and 1% non-essential amino acids during bovine
oocyte culture on subsequent in-vitro fertilization. There were no significant
differences in penetration rates among the four groups.
Table IV shows the effect of 2% essential and 1%
non-essential amino acids during bovine oocyte maturation on subsequent embryo
development. The oocyte cleavage rates were not different in the four groups.
The embryo development to the 8-cell stage were
significantly (p<0.05) higher in groups 2 (47.9±15.1) and 4 (61.2± 9) than
in the control (35.8±12.6) and group 3 (29.2±10.1).
The blastocyst rates in groups 2 (21.5±11.0) and 4
(23.6±14.0) were significantly (p<0.05) higher than in the control (2.2±4.4)
and group 3 (8.4±9.8). There were no differences in hatched blastocyst rates
among the four groups.
Table II. In-vitro maturation of bovine oocytes were cultured in
basic IVM-medium supplemented with 2% essential and 1% non-essential amino
acids (5 replicates).
Treatment
|
No. of oocytes
|
% of oocytes at the meiosis stages(Mean±SE)
|
|
|
GV
|
Metaphase-I
|
Metaphase-II
|
Control
|
92
|
1.7 ± 3.7
|
17.8 ± 7.4
|
80.5 ± 7.7
|
EAA٭
|
90
|
2.1 ± 4.7
|
16.8 ± 6.9
|
81.1 ± 7.2
|
NEAA٭٭
|
93
|
0.0 ± 0.0
|
17.7 ± 9.1
|
82.3 ± 9.1
|
EAA + NEAA
|
97
|
0.0 ± 0.0
|
9.8 ± 7.1
|
90.2 ± 7.2
|
٭ EAA= Essential amino acids ٭٭ NEAA= Non-essential amino acids
Table III. Effect of IVM-medium supplemented with 2% essential
and 1% non-essential amino acids during bovine oocyte culture on subsequent in
vitro fertilization (5 replicates).
Treatment
|
No. of oocytes
|
% of oocytes penetrated (Mean±SE)
|
|
|
Fertilization
|
Polyspermy*
|
Control
|
102
|
33.7 ± 14.9
|
35.1 ± 15.5
|
EAA٭
|
107
|
37.0 ± 16.6
|
39.5 ± 22.7
|
NEAA٭٭
|
104
|
37.3 ± 13.7
|
35.8 ± 23.4
|
EAA + NEAA
|
124
|
44.5 ± 15.1
|
32.9 ± 15.4
|
* More than one sperm
penetrated into one oocyte.
٭ EAA= Essential amino acids ٭٭ NEAA= Non-essential amino acids
Table IV. Effect of 2% essential and 1% non-essential amino
acids during bovine oocyte maturation on subsequent in-vitro embryo development
(5 replicates).
Treatment
|
No. of oocytes
|
% of oocytes cleaved (Mean±SE)
|
% of embryos developed to (Mean±SE)
|
|
|
|
8-cell stage
|
Blastocyst
|
Hatched B *
|
Control
|
100
|
79.4 ± 7.3
|
35.8 ± 12.6 a
|
2.2 ± 4.4 a
|
1.1 ± 2.2 a
|
EAA٭
|
100
|
84.6 ± 6.8
|
47.9 ± 15.1 b
|
21.5 ± 11.0 b
|
3.0 ± 4.1 a
|
NEAA٭٭
|
103
|
77.2 ± 6.5
|
29.2 ± 10.1 a
|
8.4 ± 9.8 a
|
2.7 ± 3.3 a
|
EAA + NEAA
|
100
|
82.7 ± 5.1
|
61.2 ± 9.0 c
|
23.6 ± 14.0 b
|
4.5 ± 6.5 a
|
* Hatched blastocysts.
abc Different superscripts within column indicate
significant differences (at least P < 0.05).
٭ EAA= Essential amino acids ٭٭ NEAA= Non-essential amino acids
Discussion
One of the important factors regulating the number and
quality of oocytes maturing in vitro is the culture system used for IVM.
Culture media components and culture conditions can affect and even modulate
the meiotic regulation of mammalian oocytes (12-14). It is therefore necessary
to devise and optimize culture systems that take into account all the factors
essential for the completion of oocyte maturation in vitro.
The present study showed that there were not
significant differences in oocyte maturation rates (table II) and penetration
rates (table III) among the four groups. These results were similar to the
previous results of by lim et al. (1999), who have been studied the effects of
carbohydrates and amino acids on the maturation and fertilization of bovine
oocytes. They have shown that the addition of glucose to simply defined medium
significantly enhanced oocyte maturation to the metaphaseII stage, but the
addition of EAA and NEAA to basic medium supplement with glucose did not
further improve in vitro maturation or in vitro fertilization of bovine
oocytes. Also, they have suggested that the exogenous carbohydrates and amino
acids are prerequisites for the maturation and fertilization of bovine oocytes
in vitro, glucose alone promotes the nuclear maturation of oocytes, whereas
amino acids aid the pronuclear formation of fertilized oocytes (15). Because in
our study, glucose and pyruvate were the components of the basic medium in all
four groups, probably due to their presences, maturation rates were not
different after the addition of EAA or NEAA or both. Downs and Hudson (2000)
have shown when glucose was added to pyruvate-containing cultures in mouse
oocyte, the combination of 1mM pyruvate/5.5mM glucose was most effective in
supporting maturation. The positive effect of glucose was in part attributed to
stimulation of glycolysis and increased production of pyruvate (16). Kerisher
and Bavister (17) study in cattle and Zheng et al. (18) study in rhesus monkeys
have shown that the addition of glucose to maturation media improves the
resumption of meiosis, embryo cleavage, morulae and blastocyst rates. Also, our
findings support a previous report of effects of amino acids on pig oocytes
maturation in vitro (19).
Studies on several mammalians species, including
the rabbit (20), hamster (21, 22), mouse (23), sheep (24), cattle (25) and
rhesus monkey (18) have revealed that amino acids can stimulate both oocyte
maturation and embryo development in culture.
In vivo, the mammalian embryo is exposed to
significant levels of amino acids (EAA and NEAA) in oviduct and uterine fluids
(6,26). It has been known that amino acids are transferred to the oocytes by
the action of gap junction between cumulus cells and oocytes (9). Specific
amino acid transporters are present on the membranes of oocytes and embryos and
a supply of amino acids for protein synthesis is essential for normal embryo
growth (27). It is generally accepted that mRNA and protein molecules synthesized
during oocyte growth, maturation and early embryo development is driven by mRNA
and protein stored in the oocytes (28). The embryonic genome turns on during
the 2cell stage in mice (29), the 4cell stage in rabbits (28), and the
816cell stage in bovine (30) embryos, leading to quantitative and qualitative
changes in protein synthesis, an increase in metabolic activity and the uptake
of carbohydrates. Amino acid supplementation of oocyte maturation media was
associated with enhanced developmental frequencies, increased blastocyst cell
number, and elevated oocyte maternal mRNA levels compared with defined media
without amino acids (31). The uptake and incorporation of amino acids by
embryos increased from the zygote to the blastocyst stages. Liu et al. (1996)
have demonstrated that protein synthesis must occur prior to the morula stage
for bovine embryos to develop normally into blastocysts (28). Embryos
synthesize a considerable amount of protein, particularly as they reach the
blastocyst stage (28).Therefore, the results of present study suggest that
presence of EAA and NEAA in maturation medium by the increase endogenous amino
acid pool sizes and/or de novo protein synthesis may be essential for maturation
of bovine oocytes.
In the current experiment, the percentage of
embryos that reached cleavage rate was similar for the four treatment groups,
but embryo development to the 8cell stage (68 hr post-insemination) and
blastocyst were significantly higher in groups 2 (contained EAA) and 4
(contained EAA and NEAA) than these in the Groups 3 (NEAA) and 1 (control),
(table IV). This is in agreement with the work of Liu et al. (1999), who found
that when the amino acids were excluded during the first 24 hr of culture,
rabbit embryo development proceeded compartment to the controls (28), this
indicated that the supply of endogenous amino acids available to the embryo is
sufficient during very early development (32). This is in contrast to previous
report for mouse. Amino acids have been shown to increase the cleavage rate of
mouse, producing blastocyst with higher viability (33, 36). Gardner et al. (1994)
have reported, that amino acids reduced the percentage of embryos arrested
during culture and stimulated both cleavage and hatching (26). Kim et al.
(1993) have been demonstrated that the uptake of amino acids increases from
8-cell to blastocyst stage of mouse embryos (35). Lane and Gardner (1997) have
reported that mouse embryo changed its requirements for amino acids as it
developed from the zygote to the blastocyst, development of the early cleavage
stages was stimulated by the non-essential amino acids and glutamine, but was
not effected by the essential amino acids (34). Steeves and Gardner (1994) have
revealed not only that the bovine embryos has a requirement for amino acids,
but also that amino acids have both a temporal and differential effect during
development from the 1-to-2-cell zygote to the blastocyst. They suggest that
the requirement for amino acids changes according to the developmental stage of
embryos and the metabolic requirements are different in different developmental
stage (30). Koo et al. (1997) reported that the addition of amino acids to NCSU
23 enhanced in vitro development of 1-to 2cell stage porcine embryos to the
hatching stage, because events related to protein synthesis in the hatching
process that occur prior to morula formation may be dependent upon the
availability of certain amino acids (1).
In our experiments EAA alone tended to increase
blastocyst development, while when it combined with NEAA, blastocyst production
and hatching were superior to the results using other treatments (table IV).Our results confirm Rosenkrans et al. findings (37). This is in contrast to
previous reports for hamster embryo (38), which shows that EAA alone tended to
depress blastocyst development, but similar with reports on addition of EAA and
NEAA in culture medium for mouse (23). Gardner et al. (1994) have shown that
all Eagle's amino acids significantly increase blastocyst formation, hatching
and cell number in sheep (24). Eagle's essential amino acids were inhibitory
when present before the 8-cell stage, but promoted blastocyst development and
cell number when present after the 8-cell stage (36). During development from
the 8-cell stage to the blastocyst, the non-essential amino acids and glutamine
stimulated blastocyst formation and hatching, while the essential amino acids
increased blastocyst cell number and differentiation of cells into the inner
cell mass (30). Gardner and lane (1993) have demonstrated that the inclusion of
EAA in the medium had no effect on mouse blastocyst hatching, in contrast, when
all of Eagle's amino acids were present (23), hatching was significantly
increased and further increases in the hatching rates were observed when only
the non-essential amino acids, with or without glutamine, were present. This
discrepancy might be due to a species-specific or stage-specific requirement
for amino acids during oocyte maturation and embryo development.
Conclusion
In conclusion, bovine oocytes can be successfully
developed in a chemically defined, protein free medium supplemented with EAA
and NEAA, from maturation through fertilization and culture. These data
indicate that the presence of amino acids, especially essential amino acids, in
the maturation medium is beneficial to oocyte cytoplasmic maturation and
subsequent early embryonic development in vitro. However, additional research
is needed to compare protein synthesis in oocyte cytoplasm among each group and
to compare the changes in poly (A) tail length of maternal transcripts in each
group.
Acknowledgement
The
authors would like to thank Jin-tea Chung and Ahmad-Kamal Abduljalil for their
technical assistance.
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