ORIGINAL RESEARCH

Analysis of apoptosis and expression ofgenes related to apoptosis in culturesof follicles derived from vitrified andnon-vitrified ovariesTahereh Mazoochi1, Mojdeh Salehnia1,5, Shahram Pourbeiranvand1,Mehdi Forouzandeh2, Seyed Javad Mowla3, and Ebrahim Hajizadeh4

1Department of Anatomy, Tarbiat Modares University, Tehran, Iran 2Department of Biotechnology, Tarbiat Modares University, Tehran, Iran3Department of Genetic, Tarbiat Modares University, Tehran, Iran 4Department of Biostatistic, Tarbiat Modares University, Tehran, Iran

5Corresponding author. Department of Anatomy, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran. Tel: þ98-21-88011001;Fax: þ98-21-88013030; E-mail: mogdeh@dr.com, salehnim@modares.ac.ir

The aim of this study was to evaluate the incidence of apoptosis after in vitro culture of isolated follicles derived from vitrified and non-vitrifiedovaries. Mouse ovaries were vitrified and their pre-antral follicles were mechanically isolated and cultured for 10 days. Growth and survivalrates of the follicles were assessed during the culture period and the ultrastructure of the follicles was studied. The expression of p53, Bcl-2,Bax, Fas, FasL and survivin were analyzed by real-time RT–PCR in different follicular developmental stages. The percentages of apoptotic andnecrotic cells were determined using a fluorescein-activated cell sorting (FACS) technique. There were no differences between the growthand survival rates of follicles in the vitrified and non-vitrified groups. All of the evaluated genes were expressed in the pre-antral, largepre-antral and antral follicles in both groups, except Fas mRNA, which was not expressed in the pre-antral follicles. The expression ofp53, Bcl2, Bax and FasL mRNA was similar in vitrified and non-vitrified groups; however, Fas mRNAs were more strongly expressed inthe antral follicles of the vitrified group than of the control group (P , 0.05). The expression of survivin 140 was lower in the antral folliclesof the vitrified group than of the control group (P , 0.05). FACS analysis showed that the percentage of intact cells was lower in the vitrifiedgroup than in the non-vitrified group (P , 0.05). This study demonstrated no signs of apoptosis ultrastructurally in cultured follicles; however,vitrification was shown to affect the expression of some genes related to apoptosis.

Key words: Apoptosis / Gene expression / Follicular development / In vitro maturation / Vitrification

IntroductionIn vitro culture and maturation of immature ovarian follicles is an excel-lent model to study the development of follicles and the mechanism offolliculogenesis at the genetic level (Liu et al., 2001). Also, in vitroculture and maturation of immature ovarian follicles is an alternativefor preserving the fertility potential of young persons using cryopre-served ovarian tissue.

In the literature, it has been shown that pre-antral follicles can besuccessfully cultured and matured from fresh samples or frozensamples after using slow-cooled or vitrification techniques (Cortvrindtet al., 1996, 1998; Newton and Illingworth, 2001; Liu et al., 2002;Segino et al., 2005; Haidari et al., 2006, 2008). Newton and Illingworth(2001) reported the in vitro growth of murine-isolated follicles fromcryopreserved ovarian tissue using a slow-freezing method;however, the development and survival rate of the follicles was low

in culture. Segino et al. (2005) showed that pre-antral follicles isolatedfrom vitrified mouse ovarian tissues developed more slowly than thosefrom fresh samples. Also, recent experiments in our laboratory haveshown that isolated follicles derived from vitrified, warmed ovariesproduce mature oocytes, and the ultrastructure, growth and matu-ration observed were similar in vitrified and non-vitrified samples(Haidari et al., 2008). Thus the cryopreserved follicles have the poten-tial to develop in vitro; however, the developmental rate is lower thanthat from fresh ovarian tissue.

Apoptosis is a form of programmed cell death and has been impli-cated in a spectrum of processes associated with normal functions ofthe ovary and follicular development, such as atresia and corpusluteum regression (Tilly, 1996; Kim et al., 1999; Hussein, 2005; Slotet al., 2006). Also, apoptosis may be involved in follicular damageduring freezing and thawing of the ovary (Demirci et al., 2002;Rimon et al., 2005).

& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.org

Molecular Human Reproduction, Vol.15, No.3 pp. 155–164, 2009

Advanced Access publication on January 19, 2009 doi:10.1093/molehr/gap002

There are controversial reports about the effects of cryopreservationon the incidence of apoptosis in ovarian tissue after thawing (Demirciet al., 2002; Bedaiwy and Hossein, 2004; Rimon et al., 2005; Tirelliet al., 2005; Hussein et al., 2006; Mazoochi et al., 2008). Tirelli et al.(2005) observed a significant increase in apoptosis of sow granulosacells that were frozen slowly. They suggested the increase in apoptosiswas probably caused by physical alterations due to low temperature,high salt concentration and impairment of antioxidant metabolism.Similar results were reported by Rimon et al. (2005), who demon-strated a higher incidence of apoptosis in frozen–thawed humanovarian tissue compared with fresh ovarian tissue.

In contrast, other investigations and our previous report haveshown that the incidence of apoptosis in cryopreserved tissue wasnot significantly different from fresh controls (Demirci et al., 2002;Bedaiwy and Hossein, 2004; Hussein et al., 2006; Mazoochi et al.,2008).

Despite these studies, there has been very limited attention given tothe evaluation of the incidence of apoptosis after in vitro maturation offollicles, especially in cryopreserved samples (Choi et al., 2007).

Choi et al. (2007) evaluated the proportion of apoptotic and necro-tic areas in fresh and slow-frozen and vitrified cattle ovaries 1 and 5days after culture. Their data showed that the death of ovarian cellswas significantly increased in the slow-frozen and vitrified ovaries com-pared with the fresh ovaries on Day 1; however, there was no differ-ence on Day 5 of culture.

Haidari et al. (2008) showed no significant differences between thesurvival rates of vitrified and non-vitrified, cultured, isolated follicles,and they did not observe any ultrastructural changes between vitrifiedand non-vitrified samples.

It has been demonstrated that several pro- and anti-apoptotic genesare involved in the survival of follicles, such as p53, Bcl-2, Bax, Fas, FasLand survivin (Tilly et al., 1995; Kim et al., 1998; Kugu et al., 1998; Hsuand Hsueh, 2000; Vickers et al., 2000; Slot et al., 2006; Fujino et al.,2008). Also the importance of these genes and their roles in atresiaand follicular apoptosis using mouse knockout models have beenshown in several reports (Bergeron et al., 1998; Matikainen et al.,2001; Pru et al., 2008).

The p53 protein is an antiproliferative transcription factor that con-trols genomic integrity by inducing cell cycle arrest or apoptosis. Thisprotein is expressed in the apoptotic granulosa cells of atretic follicles(Tilly, 1996).

Bcl-2 is a survival molecule that resides in the nuclear envelope andmitochondria. Its expression is found in the granulosa cells of bothfetal and adult ovaries (Tilly, 1998; Hussein, 2005; Hussein et al.,2006). Choi et al. (2004) showed a correlation between decreasesin the level of Bcl-2 mRNA with the incidence of apoptosis in isolatedgranulosa cells which were cultured under different hormonal treat-ment conditions. They considered Bcl-2 to have a critical role in inhi-biting the granulosa cell apoptosis pathway.

Bax is a pro-apoptotic protein involved in granulosa cell apoptosis(Tilly et al., 1995) and is an important regulator of follicle growth,but is dispensable for follicle atresia in mice, and a defect in folliculo-genesis was shown following Bax deletion (Greenfeld et al., 2007).

In addition to Bcl-2 family members, the Fas system is significantlyinvolved in apoptosis in the ovary (Kugu et al., 1998; Kim et al.,1999; Slot et al., 2006). Fas is a transmembrane receptorthat belongs to the tumor necrosis factor/nerve growth factor

family. It induces apoptosis when bound by Fas ligand (FasL), a typeII transmembrane protein (Krammer, 1999). The expression of Fasand FasL and their functions in the ovary have been shown (Tillyet al., 1995; Sakamaki et al., 1997; Kim et al., 1998, 1999; Tilly,1998; Krammer, 1999; Hsu and Hsueh, 2000; Vickers et al., 2000;Porter et al., 2001; Choi et al., 2004; Fujino et al., 2008; Manabeet al., 2008). Porter et al. (2001) showed higher concentrations ofFas and FasL in bovine atretic follicular cells than in healthy follicles.

Survivin is one member of the apoptosis inhibitor protein family thathas been shown to bind and inhibit the cell death effectors, caspase-3and -7 (Shin et al., 2001). It has recently been demonstrated in humansand mice (Kobayashi et al., 1999). Survivin acts in granulosa cells as abifunctional protein associated with the regulation of the cell cycle andinhibition of apoptosis (Johnson and Bridgham, 2002).

However, in the literature, there have been no reports regardingthe evaluation of the apoptotic-related genes expressed afterovarian vitrification or in in vitro culture and maturation of vitrifiedsamples.

Thus, the present study was the first attempt to evaluate theexpression of these pro- and anti-apoptotic genes (p53, Bcl-2, Bax,Fas, FasL and survivin) in isolated and cultured follicles in different devel-opmental stages from vitrified-warmed and non-vitrified ovaries. Fur-thermore, analysis of the apoptotic and necrotic cells using thefluorescein-activated cell sorting (FACS) technique in antral follicles10 days after culturing was performed.

Materials and Methods

Animals and ovarian tissueForty female 12–14-day-old National Medical Research Institute (NMRI)mice were cared for and used according to the Tarbiat Modares UniversityGuide for the Care and Use of Laboratory Animals. They were sacrificed bycervical dislocation and their ovaries (�2 mm3) were dissected free offat and mesentery and collected for the following experiments. Theovaries of immature mice were used for this study because they containa homogeneous population of pre-antral follicles, and in vivo apoptosis isminimal in these follicles.

Experimental designThe collected ovaries were categorized into vitrified and non-vitrifiedgroups. One ovary from each mouse was used randomly for the vitrifica-tion procedure and the other was reserved as the non-vitrified (fresh)control group.

Vitrification and warmingThe vitrification procedure was based on the method used previously inour laboratory (Salehnia et al., 2002). Briefly, ovarian tissues were equili-brated in vitrification medium containing 40% ethylene glycol (v/v), 30%Ficoll 70 (w/v) and 1 M sucrose supplemented with bovine serumalbumin (EGFS40) for 5 min at room temperature. Ovaries were thenplaced in 0.5 ml plastic straws with a minimum volume of the vitrificationmedium, placed on nitrogen vapor for 30 s, and then plunged into liquidnitrogen and maintained there for 1 week. Vitrified ovaries werewarmed at room temperature and then placed in a 258C water bath for20 s. The contents of each straw were expelled into 1 ml of descendingconcentrations of sucrose (1, 0.5 and 0.25 M) at room temperature for5 min.

156 Mazoochi et al.

Vitrified, warmed ovaries were equilibrated for 30 min in a-minimalessential medium (a-MEM; Gibco, Grand Island, NY, USA) supplementedwith 10% fetal bovine serum (FBS; Gibco) before follicle isolation (Haidariet al., 2006).

Isolation of pre-antral folliclesNon-vitrified and vitrified, warmed ovaries (n ¼ 5 for each group) wereplaced in 50 ml microdroplets of a-MEM. The pre-antral follicles, 100–130 mm in diameter, were mechanically isolated from the ovaries using28-gauge syringe needles under a stereomicroscope and were transferredto new microdroplets (20 ml) of medium. Only follicles containing layers ofmembrane-enclosed granulosa cells with a centrally located, healthy,visible oocyte and superficial thecal cells were selected. Generally, 15–20 pre-antral follicles were isolated from either non-vitrified or vitrified,warmed ovaries.

Toxicity testA toxicity test was performed to evaluate the effects of the vitrification sol-ution on the viability of mouse ovarian follicles. Briefly, the ovaries (n ¼ 5)were exposed to the cryoprotectant solution and passed through all stagesof the vitrification procedure, except for plunging into liquid nitrogen. Afterdehydration, the cryoprotectant was immediately removed from the tissueby transferring the ovaries into descending sucrose solutions for 5 min atroom temperature (Salehnia et al., 2002). The isolation and collection offollicles were done as described before (Haidari et al., 2006).

Evaluation of the ovarian follicular viabilityusing trypan blueThe survival rate of isolated pre-antral follicles (n , 82) derived from vitri-fied, non-vitrified and toxicity-tested groups was determined using 0.4%trypan blue (Sigma, St Louis, MO, USA) and examined under an invertedmicroscope. The follicles were scored as surviving or degenerated; degen-erated follicles stained blue, and surviving follicles were unstained (Fauqueet al., 2007).

In vitro culture of pre-antral folliclesMechanically isolated pre-antral follicles from the non-vitrified and vitrified,warmed ovaries were cultured individually in 20 ml droplets of culturemedium overlaid with mineral oil in 60 mm Petri dishes in a humidifiedatmosphere of 5% CO2 in air at 378C for 10 days. The culture mediumconsisted of a-MEM supplemented with 5% FBS (Gibco), 100 mIU/mlrecombinant follicle-stimulating hormone (rFSH or Gonal-f; Serono,Geneva, Switzerland), 1% ITS (5 mg/ml insulin, 5 mg/ml transferrin,5 ng/ml selenium; Sigma), 20 ng/ml murine recombinant epidermalgrowth factor (Sigma), 100 IU/ml penicillin and 100 mg/ml streptomycin.Media were changed by refreshing one-half (10 ml) of the medium everyother day (Haidari et al., 2006).

Assessment of follicular survival and growthThe survival rate of follicles was checked under an inverted microscopeduring the culture period and was compared on the 6th and 10th daysof culture. During the culture, the follicles were considered to have sur-vived if their structure remained normal and there was close contactbetween the oocyte and the surrounding granulosa cells; the follicleswere considered to be atretic if there was disruption of intercellularcontact with the oocyte and granulosa cells (Segino et al., 2005).

The follicles and oocyte diameters were measured at �100 magnifi-cations with a pre-calibrated ocular micrometer under an inverted micro-scope (Segino et al., 2005).

Transmission electron microscopyAll chemicals were obtained from TAAB Laboratories Ltd (Berkshire, UK).The cultured, isolated follicles in the vitrified and non-vitrified groups wererandomly collected on Day 4 of culture (n ¼ 5). They were fixed in 2.5%glutaraldehyde in phosphate-buffered saline (pH 7.4) at room temperaturefor 1.5 h and post-fixed in 1% osmium tetroxide for 2 h.

After dehydration in ascending acetone concentrations, the sampleswere embedded in Epon 812 resin. The semi-thin sections were stainedwith 1% toluidine blue, and ultra-thin sections were stained with alcoholicuranyl acetate and aqueous lead citrate. The sections were then examinedusing a transmission electron microscope (Zeiss, Gottingen, Germany).

RNA extractionSince follicular atresia is initiated by apoptosis of the granulosa cells (Umaet al., 2003), the oocytes were mechanically denuded from cultured fol-licles and the remainder of the follicles were collected and pooled in vitri-fied and non-vitrified groups, as follows: (i) pre-antral follicles at thebeginning of culture (this day was considered as Day 0); (ii) large pre-antralfollicles on Day 6 of culture, and (iii) antral follicles on Day 10 of culture.

After washing the samples in PBS and centrifuging at 1000 �g for10 min, total RNA was extracted from all of the earlier-mentionedgroups using an RNeasy MiniKit (Qiagen, Valencia, CA, USA), accordingto the manufacturer’s instructions. The RNA concentration was deter-mined by spectrophotometry and adjusted to a concentration of250 ng/ml. Using oligo dT, RNA was reverse-transcribed by Moloneymurine leukemia virus (M-MLV) reverse transcriptase. Using specifiedprimers (Table I), the p53, Bcl2, Bax, Fas, FasL and survivin genes wereamplified. b2m gene was used as an internal control and its expressionwas checked between different developmental stages of follicles. We didnot observe any remarkable changes in the expression of b2m among pre-antral, large antral and antral stages.

Three murine survivin cDNA clones (40, 121 and 140) have been iso-lated, hence we used two reverse primers for the detection of thethree survivin mRNAs (Conway et al., 2000). After reverse transcription,5 ml of reverse-transcribed cDNA product was added to the PCR reactionwith 2.5 ml of 10� PCR buffer, 0.5 ml of 10 mM dNTP mix, 0.25 ml TaqDNA polymerase (Roche Molecular System, Mannheim, Germany) and1 ml of each primer. The reaction volume was made to 25 ml withH2O. The PCR reaction cycles were set as: denaturation at 958C for30 s, annealing at 568C (for Bax, Bcl-2 and survivin) and at 578C (forp53, Fas and FasL) for 45 s and elongation at 728C for 45 s for 30 cycles.

On 1.5% (w/v) agarose gel, 10 ml of each amplified product was separ-ated, stained with 1 mg/ml ethidium bromide (Sigma) and photographedunder UV light.

Real-time RT–PCRAfter extraction of total RNA, and reconfirmation of gene expression withRT–PCR, one-step real-time PCR was performed on Rotor-gene 3000(Corbett) real-time thermal cycler according to QuantiTect SYBR GreenRT–PCR kit (Qiagen; catalog no.204243). Real-time PCR study wasdone in both vitrified and non-vitrified groups for all listed genes inTable I. Prior to the quantitative analysis, optimization procedures wereperformed by running real-time PCRs with or without template to verifythe reaction conditions, including the annealing temperatures of theprimers and specific products.

For target sequence amplifications, 100 ng (5 ml) of RNA was used per25 ml reaction volume. After completing the PCR run, melt curve analysiswas used to confirm the amplified product. For each sample, the referencegene (b2M) and the target genes were amplified in the same run. Standardcurves were obtained using the logarithmic dilution series of total RNA.

Apoptosis and cultured vitrified follicles 157

Then relative quantification of target genes was determined using the Pfafflmethod.

Flow cytometryTo detect intact, necrotic and apoptotic cells, flow cytometry was per-formed. The samples were obtained from vitrified and non-vitrified iso-lated follicles on Day 10 of culture and from the same stage of folliclesin in vivo conditions. (The last group was analyzed for comparing in vivoand in vitro conditions.) The oocytes were denuded mechanically andthe remaining follicular cells were collected and pooled. The follicularcells were separated from each other with 0.25% trypsin/0.04% EDTAin PBS, followed by 2% BSA to prevent further damage during processing.The cells (106 cells/ml) were filtered through a 30-mm nylon mesh filterand incubated for 15 min in the dark at room temperature in annexin Vincubation reagent, according to the instructions in a commercial assaykit (R&D Apoptosis Detection Kit, Wiesbaden-Nordenstadt, Germany).With this technique, intact live cells (annexin V-negative/PI-negative),early apoptotic cells (annexin V-positive/PI-negative) and necrotic orlate apoptotic cells (PI-positive) were distinguished.

All FACS analyses were performed on a PAS III flow cytometer (Partec,Gorlitz, Germany) and the data were analyzed using Partec Flomax.

Statistical analysisStatistical analysis was done with SPSS 13.0 software. The survival rates offollicles and the results of flow cytometry were assessed by the x2 test,and oocyte and follicular diameters were analyzed by Student’s t-test.

The real-time RT–PCR experiments were repeated three times and theresults were compared by one-way ANOVA test and post hoc Tukeyhonest significant difference test (P � 0.05).

Results

Follicular viability after vitrificationand warming and toxicityThe survival rate of isolated follicles derived from non-vitrified, vitrifiedand toxicity-tested samples was determined using 0.4% trypan blue;the proportions of surviving isolated pre-antral follicles were 90.2%(74/82), 80.0% (68/85) and 85.8% (73/85), respectively. Therewere no significant differences in survival rates between the groups(P . 0.05).

Survival and in vitro maturation of folliclesDevelopmental characteristics of cultured follicles derived from vitri-fied and non-vitrified ovaries are summarized in Table II. The survivalrates of follicles on the 6th day of culture in the vitrified and non-vitrified groups were 72.1 and 78.6%, respectively, and the survivalrates at the end of the culture period were 66.9 and 72.6%, respect-ively; there was no significant differences between the two groups.The oocyte diameters of the isolated follicles from the non-vitrifiedand vitrified groups on the first day of culture were 53.9+2.5 and

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Table II Developmental characteristics of cultured follicles derived from vitrified and non-vitrified ovaries

Group Follicle diameter, mm Survived, n (%) Antrum formation, n (%)

0th day 2nd day 4th day 6th day 10th day

Vitrified 124.27+7.66 133.02+7.68 197.07+10.84 83 (72.1) 77 (66.9) 30 (37.6)

Non-vitrified 121.64+8.63 155.28+8.85a 201.30+11.77 92 (78.6) 85 (72.6) 37 (43.5)

The beginning day of culturing considered as Day 0.aSignificant difference to the vitrified group in the same column.

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Table I Oligonucleotide primers

Accession numbers Gene Primer sequence PCR product size (bp)

NM-011640 p53 Forward: 50-AGAGACCGCCGTACAGAAGA-30 227Reverse: 50-GCATGGGCATCCTTTAACTC-30

NM-177410 Bcl2 Forward: 50-ACCGTCGTGACTTCGCAGAG-30 240Reverse: 50-GGTGTGCAGATGCCGGTTCA-30

NM-007527 Bax Forward: 50-CGGCGAATTGGAGATGAACTG-30 160Reverse: 50-GCAAAGTAGAAGAGGGCAACC-30

NM-007987 Fas Forward: 50-GCTGCAGACATGCTGTGGATC-30 419Reverse: 50-TCACAGCCAGGAGAATCGCAG-30

NM-010177 FasL Forward: 50-TCCAGGGTGGGTCTACTTACTAC-30 200Reverse: 50-CCCTCTTACTTCTCCGTTAGGA-30

AF 115517 Survivin Forward: 50-TCGCCACCTTCAAGAACTGGCCCTTCCTGGA-30 254,144Reverse 1: 50-GTTTCAAGAATTCACTGACGGTTAGTTCTT-30

Reverse 2: 50-GGCTTCTGACAATGACAATGCTTG-30 332

NM-009735 b2m Forward: 50-TGACCGGCCTGTATGCTATC-30 316Reverse: 50-CACATGTCTCGATCCCAGTAG-30

158 Mazoochi et al.

54.2+3.6 mm, respectively, and at the end of the culture period, theoocyte diameters were 65.8+ 2.7 and 65.6+3.1 mm, respectively;the differences between the groups were not significant.

The follicular sizes in the non-vitrified and vitrified groups at thebeginning of the culture were 121.6+8.6 and 124.3+ 7.7 mm,respectively; the follicular sizes on Day 2 of culture were 155.3+8.9 and 133.0+ 7.7 mm, respectively; and the follicular sizes on Day4 were 201.3+ 11.8 and 197.1+ 10.8 mm, respectively. Significantdifferences existed between the vitrified and non-vitrified groupsonly on Day 2 of culture (P , 0.05).

Antrum formation was observed in some of follicles from Day 8onwards; however, there was no significant differences between thenon-vitrified and vitrified groups. The rates of antrum formation inthe vitrified and non-vitrified groups on 10 Day of culture were 37.6and 43.5%, respectively.

Ultrastructural observationsElectron micrographs of vitrified and non-vitrified ovaries demon-strated that the pre-antral follicles consisted of oocytes surroundedby several layers of polyhedral granulosa and flattened theca cells.

The oocytes had a euchromatin nucleus at the germinal vesiclestage. The granulosa and thecal cells had normal morphology inboth groups. The euchromatin nuclei showed peripheral aggregatesof heterochromatin and no sign of apoptosis or chromatin conden-sation. Also, the mitochondria showed a typical ultrastructure (Fig. 1).

Expression of apoptosis-related genes incultured follicles derived from vitrifiedand non-vitrified ovariesIn this study, mRNA levels of several apoptosis-associated genes (p53,Bcl2, Bax, Fas, FasL and survivin) were evaluated in three stages of cul-turing follicles (pre-antral, large pre-antral and antral) by real-timeRT–PCR. The examined apoptotic genes were expressed differently,but among these genes, survivin 121 was not detected in pre-antral,large pre-antral and antral follicles in either the vitrified or non-vitrifiedsamples. In addition, Fas mRNA was not detected in the pre-antralstage in either group (Fig. 2).

The expression of the target genes compared with the house-keeping gene in all developmental stages of the follicles is shown inFig. 3. As the results demonstrated, among the studied genes, theexpression of p53, Bcl-2, Bax, Bax/Bcl-2 and survivin 40 did notchange significantly in the pre-antral, large pre-antral and antral folliclesin both groups. On Day 10 of culture, the level of Fas mRNA in antralfollicles was significantly higher in the vitrified group compared withthe non-vitrified group, but the survivin mRNA level was significantlylower in this group in comparison with its control (P , 0.05).

Comparison of expression ofapoptosis-related genes in differentstages of follicle developmentComparison of apoptotic genes expression in different developmentalstages of follicles revealed variations (Fig. 3). The expression of p53was lower in antral follicles than at other developmental stages of fol-licles in both groups of the study and was maximum in the pre-antralstage (Fig. 3A).

The maximum and minimum expression of Bcl-2 was observed inthe pre-antral and large pre-antral follicles, respectively (Fig. 3B).There were no significant differences between Bcl-2 expression inthe pre-antral, large pre-antral and antral follicles either in the vitrifiedor in the non-vitrified groups (P . 0.05).

The expression of Bax mRNA was not significantly differentbetween the groups at the pre-antral stage and it was decreased inthe large pre-antral follicles and antral follicles (P , 0.05) in both vitri-fied and non-vitrified ovaries (Fig. 3C).

The ratio of Bax/Bcl-2 mRNA was not significantly differentbetween all follicles at different stages in both groups of the study(Fig. 3D).

Fas mRNA was not detected in the pre-antral follicles; however, itsexpression was increased during maturation towards the antral stagein vitrified groups (Fig. 3E).

The level of the FasL gene expression was low in pre-antral folliclesand increased towards antral stage in both groups of the study, but thedifferences were not significantly different (Fig. 3F).

The level of the survivin 140 gene expression was low in the largepre-antral follicles and increased in the antral stage in the non-vitrifiedgroup (P , 0.05); no significant differences between the pre-antral,large pre-antral and antral follicles in the vitrified groups wereevident (Fig. 3G).

The survivin 40 expression showed a similar pattern in both groupsand there were no significant differences in its expression at differentfollicular stages (Fig. 3H).

Flow cytometryThe results of the flow cytometry analysis of antral follicles inthe non-vitrified, vitrified and in vivo groups are summarized inTable III.

The greatest percentage of intact cells at the antral follicle stage wasobserved in the non-vitrified group (68.8+3.8%) in comparison withthe vitrified (56.5+ 5.1%) and in vivo groups (52.2+5.2%; P , 0.05).Similarly, the percentage of necrotic cells (1.4+2.2%) and late apop-totic cells (0.1+0.01%) was significantly lower in the non-vitrifiedgroup in comparison with the vitrified and in vivo control groups(P , 0.05).

The proportions of early apoptotic cells in the non-vitrified, vitrifiedand in vivo groups were 29.7+3.3, 36.4+ 3.9 and 39.9+ 6.6%,respectively, and there was no significant difference between the vitri-fied and non-vitrified samples, whereas it was significant between thein vivo groups and non-vitrified groups (P , 0.05).

DiscussionOur results showed that the survival and growth rates of cultured pre-antral follicles derived from vitrified and non-vitrified samples weresimilar and there were no statistically significant differences. Onlythe diameter of the cultured follicles in the non-vitrified group was sig-nificantly larger than the vitrified samples on Day 2 of culture;however, after that, the difference was not significant. These datasuggest that during vitrification and warming, some changes tookplace, which could cause delay in cell growth and proliferation of fol-licular cells, but the changes were reversible.

Apoptosis and cultured vitrified follicles 159

This is consistent with a lack of significant differences in IVM out-comes between cryopreserved and fresh groups of oocytes (Liuet al., 2003; Haidari et al., 2006).

There exist conflicting reports regarding the effects of cryopreser-vation on the incidence of apoptosis (Demirci et al., 2002; Bedaiwy

and Hossein, 2004; Rimon et al., 2005; Tirelli et al., 2005; Husseinet al., 2006; Mazoochi et al., 2008). In our previous work, weshowed that just after vitrification and warming of the mouseovarian tissue, no sign of apoptosis was found using TUNEL, DNAladdering and transmission electron microscopic study (Mazoochi

Figure 1 Electron micrograph of the cultured pre-antral follicles in the vitrified and non-vitrified groups. The ultrastructure of oocytes at the germ-inal stage in the control (A) and vitrified groups (B), as the figure shows the germinal vesicle (GV) had euchromatin with a prominent nuclear envelope.The ultrastructure of granulosa cells in the control group (C) and vitrified samples (D, E and F). The nucleus (N) of these cells had marginal hetero-chromatin and normal morphology. The ultrastructure shows theca (flat cell) and granulosa cells (polygonal cell) in the vitrified group (F), and mito-chondria (M) with normal morphology distributed among the cytoplasm of these cells.

160 Mazoochi et al.

et al., 2008). But Rimon et al. (2005) have shown that the ovarianfollicles in slow-cooled samples demonstrate a higher incidence ofapoptosis compared with controls. They concluded that follicularapoptosis might be a consequence of the freezing and thawing pro-cedure (Rimon et al., 2005).

However, apoptosis is a dynamic process and it may take placesome hours after cryopreservation or during the in vitro maturationperiod. In this study, our ultrastructural observations showed thaton Day 4 of culture, there were not any apoptotic changes inoocyte and follicular and thecal cells of cultured follicles derivedfrom vitrified and non-vitrified samples. In contrast, some investi-gations showed that after follicular or granulosa cells were culturedin vitro, apoptotic cell death took place (Chun et al., 1996; Yang andRajamahendran, 2000; Ikeda et al., 2003) either in cryopreserved orfresh samples. The study of Ikeda et al. (2003) demonstrated that

cumulus cells in bovine cumulus-enclosed oocytes spontaneouslyundergo apoptosis during in vitro maturation (Ikeda et al., 2003). Inagreement with these results, Yang and Rajamahendran (2000)showed that granulosa cells from small, medium and large folliclesundergo apoptosis in serum-free culture conditions, and FSH andIGF-I can inhibit apoptosis in these cells. Our explanation for thesedifferent observations may be due to the methods of cryopreservationand safety of vitrification compared with other techniques or theimpact of follicular culture media which has been used by some inves-tigators; additional studies may be needed to improve the requiredsupplements.

We have demonstrated for the first time the expression of severalapoptotic-related genes using real-time RT–PCR technique at differ-ent stages of in vitro follicular maturation in vitrified and non-vitrifiedsamples.

Our observations demonstrated no significant difference betweenthe p53 mRNA level in vitrified and non-vitrified, cultured follicles(P . 0.05). Since p53 protein controls genomic integrity by inducingcell cycle arrest or apoptosis, our results suggest genomic integrity fol-lowing vitrification-warming may be well-preserved. The vitrificationtechnique which we have used in this study had not affected thep53 expression level, nor the Bax:bcl-2 ratio in ovarian follicles.Similar results were shown by Hussein et al. (2006), in which theydemonstrated p53 expression was not altered after slow-freezing.

In our study, the Fas mRNA was not detected in pre-antral follicles.We speculate that the absence of this pro-apoptotic gene may beresponsible for the absence of signs of apoptosis in this stage of follicu-lar development. This is in agreement with the results reported by Slotet al. (2006), who did not observe Fas protein in the granulosa cells ofhealthy pre-antral follicles.

Also, we showed that the Fas and FasL mRNA levels were lowest inpre-antral follicles and increased towards the antral stage. This may becaused by an increase in the incidence of atresia in these developmen-tal stages similar to the in vivo condition. This is in agreement with pre-vious reports which showed that Fas mRNA level was increased duringporcine follicular atresia (Inoue et al., 2006), and a high incidence ofatresia was prominent in the large follicles (Yu et al., 2004). Thestudy of Kim et al. (1999) demonstrated that the Fas/FasL systemmay be involved in the apoptosis of granulosa cells in late stages of fol-licular maturation.

Little is known about the existence and the role of the survivin inovarian follicles. In the present study, we detected survivin 40 and140 transcripts in ovarian follicles across the developmental stagesand demonstrated that the expression of survivin 140 increasedtowards the antral stage in non-vitrified groups. Other forms of survi-vin did not show notable changes between different developmentalstages. This suggests a possible physiologic role for survivin 140, overother variants of survivin, in the maintenance of follicular integrityand in the protection of follicles against apoptosis. Johnson and Bridg-ham (2002) showed that highest levels of survivin mRNA during follicledevelopment occur in mitotically active granulosa cells. They con-cluded that this protein acts as a regulator of the cell cycle and aninhibitor of apoptosis.

Moreover, our data showed for the first time that on Day 10 ofculture, the level of Fas mRNA in antral follicles was significantlyhigher in the vitrified group and it contrasted with the other obser-vation that survivin mRNA level was significantly lower in this group

Figure 2 RT–PCR analysis of gene expression of p53 (227 bp),Bcl-2 (240 bp), Bax (160 bp), Fas (419 bp), FasL (200 bp), survivin140 (254 bp), survivin 40 (144 bp) and b2m (316 bp) in cultured fol-licles derived from vitrified (Vit) and non-vitrified (Non-Vit) groups indifferent developmental stages.

Apoptosis and cultured vitrified follicles 161

in comparison with non-vitrified samples (P , 0.05). As shown in theliterature, survivin directly interacts with several caspases to inhibitapoptosis (Ambrosini et al., 1997, Shin et al., 2001), and the levelsof survivin mRNA expression decrease during the stages of folliclesatresia (Johnson and Bridgham, 2002). We speculated that a signifi-cant increase in the expression of survivin could result in the survivalof the follicles in non-vitrified samples during in vitro maturation.However, the decrease in the expression rate of this gene and theincrease in the expression of Fas gene in the vitrified group couldshow the impact of vitrification on transcription of these genes.

However, we did not observe any noticeable ultrastructuralchanges in the follicular cells after vitrification and culture. Comp-lementary expression studies at the post-translational level usingappropriate techniques such as western blot would be required toassess whether the gene expression changes observed here have afunctional effect.

In agreement with our findings, Liu et al. (2003) reported that cryo-preservation induces the expression of heat shock proteins,DNA-damage-inducible protein 45 and death-related apoptosisgenes including Fas and Fas-ligand.

Figure 3 The ratio of genes expression of p53 (A), Bcl-2 (B), Bax (C) to b2m and the ratio of Bax to Bcl-2 (D), the ratio of Fas (E), FasL (F), survivin140 (G), survivin 40 (H) to b2m using real-time RT–PCR in cultured follicles derived from vitrified and non-vitrified groups in different developmentalstages. (a) Significant differences with non-vitrified samples in the same follicular development (P , 0.05). (b) Significant differences with pre-antralfollicles in the same group of study (non-vitrified or vitrified samples; P , 0.05). (c) Significant differences with large pre-antral follicles in the samegroup of study (non-vitrified or vitrified samples; P , 0.05).

162 Mazoochi et al.

FACS analysis showed that the percentage of intact cells wassignificantly higher in the non-vitrified group compared with thevitrified group. We speculate that the vitrification may affect theintegrity of the follicular cells, which was not reflected in theirmorphology. This suggestion is in agreement with our findings ongene expression obtained by real-time RT–PCR. However, significantdifferences were not seen in early apoptotic cell populations betweenthe vitrified and non-vitrified groups. In this regard, our previousresearch showed that the developmental and survival rates ofcultured, isolated follicles derived from vitrified and non-vitrifiedsamples were almost the same and there were no significantdifferences between their growth and maturation rates in MII stages(Haidari et al., 2008).

Comparison between the in vitro (vitrified and non-vitrified) and invivo conditions revealed that the percentage of intact cells was signifi-cantly lower in in vivo follicles (P , 0.05). This may be due to the highlevel of some growth factors and hormones, such as FSHs, which havebeen used as supplementary factors in the culture media. It is knownthat FSH and epidermal growth factor act as survival factors (Chunet al., 1996; Kim et al., 1999; Yang and Rajamahendran, 2000; Yuet al., 2004), and during in vitro culture, these factors were added tothe media in supraphysiological levels, thus there was less competitionbetween follicles to gain this factor in vitro compared with the situationin vivo.

In conclusion, the present study indicated that no signs of apoptosiswere found by morphological and ultrastructural studies in the vitri-fied, cultured pre-antral follicles. However, real-time RT–PCRdemonstrated that the vitrification affected the expression of somegenes related to apoptosis. Additional studies at the protein levelare required to confirm these changes.

AcknowledgementsThe authors are grateful to Dr K. Haidari for electron microscopytechnical assistance and Mrs Makolati for real-time RT–PCR technicalassistance.

FundingThis work was supported by a grant from Tarbiat Modares University.

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Table III The flow cytometry analysis of cultured follicles at antral stage derived from vitrified, non-vitrified and freshcontrol groups using annexin V and PI staining

Group Non-intact cells Intact cellsmean+++++SD (%)Early apoptotic

mean+++++SD (%)Late apoptotic ornecrotic mean+++++SD (%)

Necroticmean+++++SD (%)

Non-vitrified 29.7+3.3 0.1+0.01 1.4+2.2 68.8+3.8

Vitrified 36.4+3.9 2.1+0.3a 5.0+1.1a 56.5+5.1a

In vivo 39.9+6.6a 4+0.8a,b 3.9+0.7a,b 52.2+5.2a,b

aSignificant differences within the same column compared with non-vitrified group (P , 0.05).bSignificant differences within the same column compared with vitrified group (P , 0.05).

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Submitted on May 26, 2008; resubmitted on December 27, 2008; accepted onJanuary 14, 2009

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