Mahboobeh Amoushahi1, Mojdeh Salehnia2, Seyed Javad Mowla3, Nassim Ghorbanmehr4. 1. Department of Anatomy, Tarbiat Modares University, Tehran, Iran. 2. Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: salehnim@modares.ac.ir. 3. Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. 4. Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.
Ovarian tissue cryopreservation is a good
alternative method for preserving fertility in
women suffering from premature ovarian failure
(POF) or cancer that requires chemotherapy or
radiotherapy (1). One method for the ovarian tissue
cryopreservation of mammals, which has been
widely studied, is vitrification (2). This method
consists of a physical procedure, in which a high
concentrated cryoprotectants applied without
formation of any ice crystals to vitrified the living
cells (3). Several studies have been conducted to improve ovarian vitrification method (4-7), while
the successful results have been obtained on cow
(8), mice (9, 10), rat (11) and human (12).One of the methods to preserve ovarian fertility
after cryopreservation is in vitro maturation of
ovarian follicles, which has been applied in the
recent investigations (13-15). Their results are
different, but impact of vitrification on ovarian
tissue is still ambiguous (16-18). It has been
suggested that vitrification affects follicular
development, leads to DNA damage and
causes loss of some cytoplasmic mRNA (19).
Moreover, a group of studies have reported
some controversial results regarding the effects
of vitrification methods on gene expression
patterns of follicles in mammals (20-22). Fatehi
et al. (23) have showed that the expression of
some genes related to folliculogenesis (Bmp15,
Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2) had
no effects on vitrified follicles cultured in a
two-dimensional system as compared with the
fresh control group. Asadzadeh et al. (24) have
recently reported that expression of Timp-2 and
Mmp-2 genes were altered after in vitro culture
of isolated follicles derived from vitrified
mouse ovarian tissue. Sampaio da Silva et al.
(25) have recently showed that the proliferation
of granulosa cells in developing follicles
decreased after vitrification of ovarian tissue
by changing in the gene expression of Cx43 in
secondary follicles.Therefore, study of the pattern of follicular
gene expression in short- and long-term
culture may be a useful method to assess the
impact of vitrification on the development and
maturation of follicles and oocytes. A number
of studied have indicated that in vitro culture of
ovarian tissue may influence on the follicular
development through some alterations in the
expression of genes involved in folliculogenesis
(23, 26). Ovarian follicular development is
a complex process (26) and the expression
of different genes, such as proliferating cell
nuclear antigen (Pcna), follicle-stimulating
hormone receptor (Fshr), P450, and Cyp17a1,
in this process are of importance, since changes
in the expression of these genes may have
some impacts on the process of follicle, oocyte
or embryo development (23). PCNA is one of
the essential regulators of cell cycle with a
molecular weight of 36 kDa. Due to being a
cofactor for DNA polymerase delta at S phase
cycle, PCNA plays a significant role in repair
of damaged DNA. PCNA is also considered as
a very important marker in cell proliferation
due to strong binding to cyclin D at S phase. A
number of studies have reported the expression
of this gene in the ovaries of different species
during follicular development such as pigs
(27), cows (28), baboons (29) and mice (30).
Expression of Pcna increases in G and S phases,
while it decreases in M phase. Cyp17a1 is a
critical factor that involves in folliculogenesis
and biosynthesis of steroid hormones (31, 32).
FSHR as an internal membrane cell surface
receptor is expressed on the granulosa cells of
secondary follicles and plays an essential role
in the transmission of follicles to the antral
stage (33, 34).Given the importance of Pcns, Fshr and
Cyp17a1 genes in the development of ovarian
follicles and due to lack of necessary information
regarding their changes throughout the in vitro
culture of vitrified ovaries, this study aimed
to evaluate the expression of these genes in
follicles after vitrification of mouse ovarian
tissue using a two-step in vitro culture.
Materials and Methods
All chemicals were obtained from Sigma Aldrich
(Munich, Germany) except mentioned otherwise.
In this experimental study, a group of neonate
(n=30) and adult NMRI male mice (n=10) were
kept in a cycle of 14 hours light and 10 hours dark,
at 20-24ºC and 40-50% humidity. Approval for
this study was obtained from the Ethics Committee
for Animal Research of the Tarbiat Modares
University (Ref No: 52/1637).
Experimental design
After collection of neonate female miceovaries
(n=60), they were randomly divided into two
studied groups as follows: vitrified and non-vitrified
groups. Then some of the tissues in both groups
were considered as non-cultured and subjected
to morphological evaluation and the others were
cultured. The whole ovaries were cultured on
the inserts for 7 days. In vitrified group (n=15),
the morphology of ovaries and the percentage of
follicles were evaluated with histological studies.In non-vitrified group (n=15), their preantral
follicles were isolated and cultured in a threedimensional
culture system for 12 days. Then,
the rates of fertilization and embryo development
were evaluated in collected metaphase (M) II
oocytes. The expression of several genes related
to folliculogenesis was evaluated using real time
reverse transcription-polymerase chain reaction
(RT-PCR) at the end of culture in collected
follicles.
Ovarian collection
To collect ovaries, neonate female mice were
killed by cervical dislocation, removed their
ovaries with surrounding tissues, dissected, and
washed with alpha-Minimal Essential Medium
(α-MEM, Gibco, UK) supplemented with 5% fetal
bovine serum (FBS, Gibco, UK).
Vitrification and warming
The ovaries (n=25) were vitrified as previously
described (35). Briefly, the ovaries were transferred
into vitrification solution, EFS40, containing 40%
(v/v) ethylene glycol, 30% (w/v) Ficoll 70%
(w/v), and 1 M sucrose supplemented with 10%
bovine serum albomin (BSA) for 5 minutes at
room temperature. Then, they were loaded onto
the Cryolock and plunged into liquid nitrogen for
1 week. Afterward, the Cryolock was sequentially
placed into 1, 0.5 and 0.25 M sucrose solutions
containing 10% BSA in α-MEM medium for 5
minutes at room temperature. After warming,
the ovaries were incubated for 1 hour in α-MEM
medium supplemented with 5% FBS under
mineral oil at 37˚C in a humidified atmosphere
of 5% CO2-95% air. Then some of these tissues
were considered as non-cultured and the others as
cultured groups.
Ovarian culture
The ovaries in both groups were cultured on
Millicell-CM inserts (pore size of 0.4-μm, 30 mm
diameter, Millipore Corp., Germany) in the 24-
well plates at 37˚C and in a humidified atmosphere
of 5% CO2-95% air for 1 week. The culture
medium was α-MEM medium supplemented with
5% FBS, 1% insulin-transferrin-selenium medium
(ITS, Gibco, UK), and 100 mIU/ml recombinant
follicle stimulating hormone (rFSH or Gonal-F,
Serono, Switzerland). Half of the medium in each
well were replaced with fresh medium every other
day during culture period.
Histological evaluation
At the first and last days of culture period (7 days),
the ovaries in each group (n=5/each group) were
fixed in Bouin’s solution, processed and embedded
in paraffin wax. Then, they were sectioned serially
into 5 μm-thick slices, mounted on a glass slide,
and stained with hematoxylin and eosin (H&E).
Every 5th section of each ovary was studied for
counting the follicles. Classification of follicles
was described previously (36). Briefly, primordial,
primary and preantral follicles were considered as
those had flattened granulosa cells surrounding the
oocyte, one layer of cuboidal granulosa cells, and
two or more layers of cuboidal granulosa cells,
respectively. To prevent duplicate count, only
follicles with one nucleus were counted. Follicles
with an intact oocyte and organized granulosa
cells were considered as normal follicles, whereas
degenerated follicles contained piknotic oocyte
nuclei, shrunken ooplasm, and/or disorganized
granulosa cells.
Surface ovarian area
The images of follicles of both studied groups
were prepared using an invert microscope with an
attached DP11 digital camera (Olympus, Japan).
Surface of ovary (n=5/each group) was calculated
based on μm² using Digimizer software system
(MedCalc Software, Belgium).
Follicle isolation and assessment of ovarian
follicle viability by trypan blue staining
Preantral follicles, 140-150 μm in diameter,
were mechanically isolated from cultured ovaries
in both groups (n=170/each group) by a 29 gauge
needles under a stereomicroscope (Olympus,
Japan). Selected preantral follicles had central
oocyte, 2-3 layers of granulosa cells and a thin
layer of theca cells. Some of isolated preantral
follicles from the cultured ovaries in both groups
(n=20/each group) were stained using trypan blue
(0.4%). The survived follicles were those that
were not stained, whereas the damaged follicles
were those that were stained moderately blue. The
other isolated follicles were cultured using a threedimensional
culture system.
The encapsulation and culture of isolated
preantral follicles
The isolated preantral follicles (n=150/each
group) were encapsulated in sodium alginate as
defined previously. Briefly, the follicles were
cultured in α-MEM medium supplemented with
FBS, rFSH, ITS, penicillin, and streptomycin under
mineral oil at 37˚C in a humidified atmosphere of
5% CO2-95% air for 12 day. The half of media was
changed every other day during culture period
Assessment of follicular diameter and
development
The morphology of cultured follicles was assessed
under an inverted microscope (Olympus, Japan)
every 48 hours during culture period. The follicles
with dark appearance were defined as degenerated
ones. The follicle diameter (n=10/each group) was
determined by precalibrated ocular micrometer
(Olympus, Japan) using an inverted microscope
(magnification: ×100) during culture period.
In vitro ovulation induction
After 12 days, ovulation induction was carried out
by adding 1.5 IU/mL human chorionic gonadotropin
hormone (hCG, Organon, Netherlands) to the culture
media. Then, some of cultured follicles were collected
and stored at -80˚C for molecular assessment (n=30/
each group), while in others cultured follicles (n=60/
each group), the released oocytes were scored as
germinal vesicle, germinal vesicle breakdown and
MII. The oocytes at MII stage were collected and
subjected to insemination and assessment of the
embryo development.
In vitro fertilization and embryo culture
In a three-dimensional culture system, after in vitro
culture of isolated follicles, the collected oocytes
at MII stage were inseminated with capacitated
spermatozoa derived from cauda epididymis of 7-8-
week old male NMRI mice (n=10) in the global
medium (Life Global, USA) supplemented with 15
mg/ml BSA for 4-6 hours. Then, the oocytes were
removed and placed into a 20-μl drop of global
medium with 5 mg/ml BSA under mineral oil at
37˚C in a humidified atmosphere of 5% CO2-95% air.
Fertilization and developmental rates of 2-cell, 4-cell,
8-cell, morula and hatching blastocyst embryos were
evaluated daily for 120 hours.
RNA extraction
Total RNA was extracted from collected follicles
in vitrified and non-vitrified groups at the end
of culture period (n=30 follicles/each group, 10
follicles for each replicate of experiments) using
RNeasy Mini Kit (Qiagen, Germany). To eliminate
any genomic DNA contamination, DNase treatment
was performed after RNA extraction. Determination
of RNA concentration was then performed using
spectrophotometry (Shimadzu, Japan), and RNA
samples were stored at -80˚C. The cDNA was
synthesized by oligo (dT) primers and reverse
transcriptase at 42˚C in 60 minutes using the cDNA
Synthesis Kit (Thermo Scientific, EU), according to
the manufacturer’s instructions, and stored at -20˚C.
Designed primers by GenBank (http://www.ncbi.
nlm.nih.gov) and Allele ID software are shown in
Table 1, and β-actin was used as housekeeping gene
in this study. The Applied Biosystem Real Time
PCR Cycler according to QuantiTect SYBR Green
RT-PCR kit (Applied Biosystems, UK) was used.
Amplification of reference and target genes was
done in the same run for each sample. The protocol
of real time RT-PCR was programmed as follows:
the holding step at 95˚C for 5 minutes, cycling
step at 95˚C for 15 seconds, 58˚C for 30 seconds,
and 72˚C for 15 seconds, which was followed by
a melt curve step at 95˚C for 15 seconds, 60˚C for
1 minute, and 95˚C for 15 seconds. The relative
quantitation for target genes was determined using
Pfaffl method. All experiments of real time RTPCR
were replicated three times.
Statistical analysis
All experiments were repeated at least three
times. Values are given as mean ± standard error
(SE). The data of follicular count, ovarian area
and gene expression in the vitrified and nonvitrified
groups were compared using paried t
test. The percent of normal follicles at different
developmental stages were analyzed by one-way
ANOVA and tukey’s HSD was used post hoc tests.
The statistical analysis was accomplished using the
Statistical Package for the Social Sciences version
21 (SPSS, SPSS Inc., USA). A value of P<0.05
was considered as statistically significant.
Results
Morphology of ovaries
The measurement values of phase contrast
morphology of non-vitrified and vitrified ovaries
on days 0, 5 and 7 of culture period are shown in
Figure 1. The follicles were grown and the anterior
surfaces of ovaries were swollen at the end of
culture period in both groups. The dark areas in the
central part of the cultured ovaries were detected in both groups; however, it was prominent in the
vitrified sample. Figure 2 shows the morphology
of vitrified and non-vitrified ovaries before and
after culture period using H&E. Growing follicles
with normal morphology were visible on day 7 of
culture period in non-vitrified and vitrified ovaries.
Moreover, degenerated follicles, especially in
central area of ovaries, were detected in both
groups. It seems that the degenerated follicles
in the vitrified samples were more than the nonvitrified
group.
Fig.1
Photomicrographs of mouse ovaries under an inverted microscope during culture period are shown. The representative figures on days
0, 5, 7 in A-C. Non-vitrified and D-F. Vitrified groups are seen respectively.
Fig.2
Photomicrographs of vitrified and non-vitrified whole mouse ovaries sections before and after 7-day culture using H&E staining. A.
Non-cultured non-vitrified mouse ovary, B. Non-vitrified cultured ovary with low magnification, C, D. with high magnification, E. Vitrified
non-cultured ovary, F. Vitrified-cultured ovary with low magnification, G, and H. with high magnification. It is noted that growing follicles
are visible on day 7 of culture in non-vitrified and vitrified ovary, while degenerated follicles, especially in central areas of ovary, are
demonstrated in both groups of study. It seems that the degenerated follicles in the vitrified samples are more than the non-vitrified
group. H&E; Hematoxylin and eosin staining method.
Designed primer sequences used for real-time reverse transcriptase- polymerase chain reaction (RT-PCR)Photomicrographs of mouseovaries under an inverted microscope during culture period are shown. The representative figures on days
0, 5, 7 in A-C. Non-vitrified and D-F. Vitrified groups are seen respectively.
Percentage of normal follicles
The percentages of normal follicles in non-vitrified
and vitrified groups before in vitro culture were 96.09
and 95.27%, while after one week in vitro culture,
they were 73.95 and 73.42%, respectively. There was
no significant difference regarding the normality rate
of follicles between vitrified and non-vitrified groups,
but it reduced in both cultured groups as compared
with non-cultured samples (P<0.001). Among the
normal follicles, the proportions of follicles in noncultured
non-vitrified group at primordial, primary
and preantral stages were 92.54, 5.31, and 2.14%,
while these percentages in non-cultured vitrified group were 92.56, 5.32, and 2.12%, respectively (Table 2).
In cultured non-vitrified group, after 7 days of culture,
the percentages of follicles at primordial, primary
and preantral stages were 65.48, 10.00, and 24.50%,
while those of cultured vitrified group were 69.55,
11.18, and 18.40%, respectively. Many of follicles at
primordial stage were grown to preantral stage during
the culture period in both groups, indicating there
was significant difference regarding percentage of
preantral follicles before and after culture within each
group (P<0.001). The proportion of preantral follicles
in vitrified group was significantly lower than those of
non-vitrified group on day 7 of culture (P<0.05).
Table 2
The percentages of follicles at different developmental stages in studied groups
Group
Total
Number of normal follicles (%)
Primordial folliclen (mean% ± SE)
Primary folliclen (mean% ± SE)
Preantral folliclen (mean % ± SE)
Non-cultured non-vitrified ovaries
3404
3271 (96.09)
3027 (92.58 ± 0.81)
174 (5.27 ± 0.65)
70 (2.1 ± 0.19)
Non-cultured vitrified ovaries
2960
2820 (95.27)
2610 (92.55 ± 1.61)
150 (5.31 ± 1.36)
60 (2.12 ± 0.32)
Cultured non-vitrified ovaries
1840
1359 (73.85)
890 (65.52 ± 1.16)a
136 (9.96 ± 0.48)a
333 (24.50 ± 1.07)a
Cultured vitrified ovaries
1554
1141 ( 73.42)
802 (69.56 ± 0.23)b
129 (11.19 ± 0.58)b
210 (18.41 ± 0.33)b, c
The percentage of follicles was calculated based on the normal follicles. a; Significant difference with non-cultured non-vitrified ovaries
(P<0.001), b; Significant difference with non-cultured vitrified ovaries (P<0.001), c; Significant difference with cultured non-vitrified ovaries
(P<0.05), Real time PCR; Real time reverse transcription-polymerase chain reaction, and SE; Standard error.
Photomicrographs of vitrified and non-vitrified whole mouseovaries sections before and after 7-day culture using H&E staining. A.
Non-cultured non-vitrified mouse ovary, B. Non-vitrified cultured ovary with low magnification, C, D. with high magnification, E. Vitrified
non-cultured ovary, F. Vitrified-cultured ovary with low magnification, G, and H. with high magnification. It is noted that growing follicles
are visible on day 7 of culture in non-vitrified and vitrified ovary, while degenerated follicles, especially in central areas of ovary, are
demonstrated in both groups of study. It seems that the degenerated follicles in the vitrified samples are more than the non-vitrified
group. H&E; Hematoxylin and eosin staining method.The percentages of follicles at different developmental stages in studied groupsThe percentage of follicles was calculated based on the normal follicles. a; Significant difference with non-cultured non-vitrified ovaries
(P<0.001), b; Significant difference with non-cultured vitrified ovaries (P<0.001), c; Significant difference with cultured non-vitrified ovaries
(P<0.05), Real time PCR; Real time reverse transcription-polymerase chain reaction, and SE; Standard error.
Area of ovaries
The data of surface area analysis in cultured
ovaries is presented in Figure 3. The mean areas
in non-vitrified and vitrified cultured ovaries
significantly increased from 53476.40 ± 568.97
and 53287.80 ± 410.44 μm² on day 0 to 297211.40
± 6671.71 and 162468.20 ± 703.78 μm² on day 7
of culture, respectively (P<0.05). Surface areas of
vitrified cultured ovaries were significantly lower
than non-vitrified cultured ovaries on day 7 of
culture (P<0.05).
Fig.3
The mean area of mouse cultured vitrified and nonvitrified
ovaries during a 7-day culture. a; There was a significant
difference in this regard between two studied groups (P<0.002)
and *; There were significant differences between days 7 and 5
with other days within each studied group (P<0.01)
Survival rate of isolated preantral follicles
Survival rates of isolated preantral follicles
from cultured non-vitrified and vitrified ovaries
after culture period were 80.35 and 79.45%,
respectively, suggesting that there was no
significant difference in this regard between two
treatment groups (P>0.05).
Diameter of isolated preantral follicles
The mean diameters of cultured follicles in nonvitrified
and vitrified ovaries groups significantly
increased from 148.40 ± 1.14 and 144.80 ± 0.83
μm on day 0 to 410 ± 7.90 and 405.80 ± 6.72
μm on day 12, respectively (Fig .4, P<0.001),
suggesting that there was no significant difference
in this regard between two treatment groups during
culture period (P>0.05).
Fig.4
Diameter of cultured isolated preantral follicles in vitrified and
non-vitrified groups. Values are given as mean ± standard deviation
(SE). a; There was significant differences between days 12 with other
days.
Developmental rate of follicles
The data related to development of follicles in
both groups are summarized in Table 3. There
was no significant difference regarding antrum
formation between cultured non-vitrified and
vitrified groups. The percentages of MII oocytes
derived from cultured preantral follicles in nonvitrified
and vitrified groups were 32.11 ± 2.39 and
30.00 ± 4.43, respectively. There were no significant
differences in terms of the developmental rate of
follicles and percent of matured oocytes reaching
MII stage between two treatment groups (P>0.05).
Table 3
The developmental and maturation rates of cultured preantral follicles
Group
Number of follicles
Survived (%)
Antrum formation (%)
Germinal vesicle (%)
MI (%)
MII (%)
Non-vitrified ovaries
102
76 (74.50)
45 (59.21)
22 (28.94)
31 (40.78)
23 (30.26)
Vitrified ovaries
136
100 (73.52)
58 (58)
29 (29)
41 (41)
30 (30)
The percentage was calculated based on the survived follicles. There was no significant difference between vitrified and non-vitrified
groups (P>0.05). M; Metaphase.
The rates of fertilization and embryo development
Fertilization rates of MII oocytes derived from
cultured preantral follicles were 78.95 and 71.66%
in cultured non-vitrified and vitrified groups,
respectively (Table 4), indicating that there was no
significant difference in this regard between two
treatment groups (P>0.05).
Table 4
Fertilization and developmental rates of metaphase II oocytes in studied groups
Group
Number of MII
Number of fertilized (%)
Number of 2-cell (%)
Number of morula (%)
Number of hatched blastocyst (%)
Cultured non-vitrified ovaries
28
21 (78.95)
16 (77.33)
10 (45.33)
6 (29.33)
Cultured vitrified ovaries
25
18 (71.66)
14 (78.66)
8 (44.66)
5 (29.00)
Gene expression analysis
The mean expression ratios of Pcna, Fshr and
Cyp17a1 to housekeeping gene in antral follicles
derived from non- vitrified ovaries were 0.15 ±
0.01, 0.33 ± 0.14, and 0.13 ± 0.02, while these
ratios in vitrified ovaries were 0.14 ± 0.01, 0.22 ±
0.05, and 0.17 ± 0.06, respectively (Fig .5). There
were no significant differences in terms of the mean
expression ratios of Pcna, Fshr and Cyp17a1 to
housekeeping gene between cultured vitrified and
non-vitrified groups (P>0.05).
Fig.5
The mean expression ratios of Pcna, Fshr and Cyp17a1
to a housekeeping gene (β-actin) by real time real time reverse
transcription-polymerase chain reaction (RT-PCR) in cultured
antral follicles on day 12 derived from whole ovarian culture.
There was no significant difference in this regard between
vitrified and non-vitrified groups (P>0.05).
The mean area of mouse cultured vitrified and nonvitrified
ovaries during a 7-day culture. a; There was a significant
difference in this regard between two studied groups (P<0.002)
and *; There were significant differences between days 7 and 5
with other days within each studied group (P<0.01)Diameter of cultured isolated preantral follicles in vitrified and
non-vitrified groups. Values are given as mean ± standard deviation
(SE). a; There was significant differences between days 12 with other
days.The developmental and maturation rates of cultured preantral folliclesThe percentage was calculated based on the survived follicles. There was no significant difference between vitrified and non-vitrified
groups (P>0.05). M; Metaphase.Fertilization and developmental rates of metaphase II oocytes in studied groupsThe mean expression ratios of Pcna, Fshr and Cyp17a1
to a housekeeping gene (β-actin) by real time real time reverse
transcription-polymerase chain reaction (RT-PCR) in cultured
antral follicles on day 12 derived from whole ovarian culture.
There was no significant difference in this regard between
vitrified and non-vitrified groups (P>0.05).
Discussion
Our findings showed that after one week in
vitro culture of neonatal mouse ovarian tissue,
there was a significant reduction in proportion of
normal follicles in both vitrified and non-vitrified
groups as compared to non-cultured tissues.
However, this effect was more prominent in the
vitrified group. It is suggested that in vitro culture
condition is needed to be improved to enhance the
follicular survival and development. Moreover,
our results indicated that the vitrified sample
was more sensitive to insufficient condition of
the culture. Similarly, in our previous study, just
after vitrification/warming, the integrity of tissue
was well preserved; however, after one week in
vitro culture, deleterious effects of vitrification on
the follicular morphology and development were
detected (37). In the present study, there were
no significant differences between the follicular
survival rate and antrum formation with the
percentage of oocyte maturation in vitrified and
non-vitrified groups at the end of two-stage culture
of ovarian tissue, suggesting that vitrification had
no negative impacts on follicular development at
morphological level after in vitro culture of isolated
follicles (steps two of in vitro culture). Moreover,
our results demonstrated that a three-dimensional
culture system provided an environment similar
to human ovary in order to preserve the spherical
shape of follicle, which is in agreement with a
group of studies using a three-dimensional culture
system for the growth of pre-antral follicles (38-
41). Abdi et al. (42) have recently showed that
a three-dimensional culture system of isolated
preantral follicles from cultured mouse ovarian
tissue provided better conditions for survival
and development of follicles as compared to the
conventional two- dimensional culture system.However, alteration in the gene expressions
related to the oocyte maturation during culture period in both groups may influence subsequent
embryo development. Results of the present study
showed that there was no significant difference in
gene expression of Cyp17a1, Fshr and Pcna in
both vitrified and non-vitrified groups after a twostep
culture that may confirm the morphological
results in both groups at the molecular level.Pcna as one of the factors in regulating
development of ovarian follicles participates in
cellular vital processes such as cell cycle control,
cell survival, replication, repair and prevention
of DNA damage; however, its exact function in
meiosis, especially in formation of primordial
follicles is still unknown. Xu et al. (30) have
reported that reduced expression of Pcna caused
a delay in transition of follicles from primordial
to primary stage, while it reduced the proliferation
of somatic cells. Choi et al. (43) have isolated and
cultured preantral follicles of 12-day old mice after
ovarian tissue vitrification. They have then reported
that in the vitrification group, the expression of
Pcna at 0 and 24 hours after culturing follicles
significantly reduced in comparison with the
control group. However, there was no significant
difference between the two groups at 48 to 72
hours after culture. Their results have showed that
proliferation of granulosa cell was delayed after
vitrification, although it improved after 48 hours
of culture period.Fshr is expressed in granulosa cells of
preantral follicles, and the reaction of this
receptor with FSH hormone leads to follicle
growth by proliferation and differentiation of
granulosa cells and the formation of antrum
(34). Cyp17a1 has a key role in synthesis of
steroid hormones. A group of studies have revealed
that suppression of Cyp17a1 decreased the levels
of progesterone, androstenedione, testosterone
and 17a-hydroxyprogesterone (17-OHP) (44-
46). Similarly, Fatehi et al. (23) cultured isolated
preantral follicles from vitrified and non- vitrified
12- day old mouse ovarian tissues by a twodimensional
culture system. Their results have
indicated that the expression of some genes related
to folliculogenesis (Bmp15, Gdf9, BmprII, Alk6,
Alk5, Has2 and Ptgs2) had not effects on vitrified
isolated and cultured follicles as compared with
the fresh control group.In another study by Shams Mofarahe et al. (47),
they have showed that there was no significant
difference in terms of expression of genes related
to folliculogenesis (FIGLA, KIT Ligand, GDF9
and FSHR) between the fresh human ovarian tissue
with vitrified- warmed samples. In contrast to our
results, Asadzadeh et al. (24) have reported that
expression of some genes related to development
of follicles (Timp-2 and Mmp-2) were altered after
vitrification of in vitro culture of mouse ovarian
tissues.Isachenko et al. (18) evaluated human ovarian
tissue after droplet vitrification method and 16
days culture in molecular level. Their findings
have indicated that after both vitrification and
culture, gene expression of GAPDH in ovarian
tissue reduced, suggesting that this difference may
be due to different methods used for vitrification or
different culture periods. Our findings demonstrated
that in spite of reduction in the percentage of
preantral follicles and ovarian surface area after
7-day culture in the vitrified group in comparison
with non-vitrified group, there was no significant
difference between the two groups in expression of
genes related to development of follicles, oocyte
maturation, fertilization and embryo development
rate. This result may be due to the damaged
follicles during the vitrification process that were
ignored from following 12-day culture. On the
other hand, by isolation of follicles, the intact and
healthy follicles or the ones with less injury during
the vitrification process were selected after culture
period. However, it is suggested that more genes
are required to be evaluated after vitrification
and culture of ovarian tissue. Furthermore, after
embryo transfer, the implantation rate and in vivo
embryo development are needed to be assessed.
Conclusion
The results of this study showed that vitrification
of ovarian tissue following in vitro culture of tissue
had negative impact on survival and development
of the follicles. However, no significant alteration
was observed in development, gene expression and
hormonal production of in vitro culture of preantal
follicles derived from vitrified ovarian tissue as
compared to the non-vitrified samples.
Table 1
Designed primer sequences used for real-time reverse transcriptase- polymerase chain reaction (RT-PCR)
Authors: Fuminori Taniguchi; John F Couse; Karina F Rodriguez; Judith M A Emmen; Donald Poirier; Kenneth S Korach Journal: FASEB J Date: 2006-12-08 Impact factor: 5.191
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