CELL PRESERVATION TECHNOLOGYVolume 4, Number 4, 2006© Mary Ann Liebert, Inc.DOI: 10.1089/cpt.2006.9998

In Vitro Culture of Cryopreserved Caprine Ovarian Tissue Pieces and Isolated Follicles

A.P.R. RODRIGUES,1 S.H.F. COSTA,1 R.R. SANTOS,1 C.A. AMORIM,2 C.M. LUCCI,2S.N. BÁO,2 J.F. NUNES,3 D. RONDINA,4 and J.R. FIGUEIREDO1

ABSTRACT

The objective of the present study was to evaluate the effects of cryopreservation of goat preantral follicles, in situand isolated, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Follicles were cryopreserved and cul-tured in vitro, and follicle viability was assessed by Trypan Blue staining before and after culture. The viabilityof follicles cryopreserved in situ using 1.5 M DMSO (64% � 5.3 and 60% � 7.4) or 3.0 M EG (58% � 3.7 and 45% �4.4) after days 1 and 5, respectively, of culture was lower (p � 0.05) than before culture (84% � 6.9). Similar resultswere obtained with isolated follicles cryopreserved in 1.5 M EG after 1 (51% � 6.5) and 5 (63% � 6.6) days of cul-ture. The viability of fresh and cryopreserved follicles in situ was similar on days 0, 1, and 5 of culture (P � 0.05).For isolated follicles, the percentage of viable fresh follicles was higher than follicles cryopreserved in EG on days1 and 5 of culture, and in DMSO on day 1 of culture. Follicle diameter was not altered during culture, both forfresh and cryopreserved follicles and when follicles were cultured in situ or isolated. In conclusion, caprine pre-antral follicles were successfully cryopreserved, especially in situ, and follicle viability after isolation was simi-lar between fresh and cryopreserved follicles after 1 day of culture, when DMSO was used.

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INTRODUCTION

THE MAMMALIAN OVARY has a great stock ofpreantral follicles, e.g., about 90% of all fol-

licles within the ovary1 that contain small im-mature oocytes in the germinal vesicle (GV)stage. Due to their small size and lack of differ-entiation, preantral follicles are less susceptiblethan antral follicles to damage induced by cry-opreservation.2–4 In fact, many studies havebeen conducted regarding cryopreservation andtransplantation of pieces of ovarian tissue in anattempt to restore fertility.5–9 Since different cul-ture systems have been tested for preantral fol-licles isolated10–12 or within ovarian tissue,13–15

cryopreservation of these follicles can be an ef-fective method to preserve female germ cells.16

Normal folliculogenesis can occur in piecesof ovarian tissue previously cryopreserved andtransplanted to the kidney capsule of immuno-deficient mice.7 Also, pregnancy was achievedin mice17 and ewes6,18 after cryopreservedovarian tissue transplant, with birth of liveyoung in the latter. Nowadays, the rate of sur-vival and growth of preantral follicles is higherafter transplantation of ovarian tissue6,19–22

than after in vitro culture of ovarian tissue orisolated follicles.12 However, in vitro culturehas advantages. For example, in women whomight use ovarian tissue cryopreservation be-

1Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles—LAMOFOPA—Ceará State University, CE,Brazil.

2Laboratory of Electron Microscopy, University of Brasília, DF, Brazil.3Laboratory of Technology of Caprine and Ovine Semen—Ceará State University, CE, Brazil.4Laboratory of Physiology and Control of Reproduction—Faculty of Veterinary, Ceará State University, Fortaleza,

CE, Brazil.

fore cancer treatments, ovarian transplant rep-resents a risk of reintroducing malignant cellsin the patient.23 This risk can be reduced oreliminated by the complete growth and matu-ration of ovarian follicles in vitro.12

Regarding isolated follicles, murine pre-antral follicles frozen/thawed and cultured invitro after isolation were fertilized and devel-oped to blastocysts.10,11 Moreover, live youngwere born after cryopreservation and in vitroculture and fertilization of mice-isolated pri-mary follicles.24,25 In domestic cats, Jewgenowet al.26 obtained 20% viable follicles after cryo-preservation after 7 days of culture. Althoughgood results have been achieved with cryop-reservation of murine and feline preantral fol-licles, there is no information about in vitro cul-ture following cryopreservation of isolatedcaprine preantral follicles.

The efficiency of dimethyl sulfoxide(DMSO), ethylene glycol (EG), propanediol(PROH), and glycerol (GLY) to cryopreservecaprine preantral follicles in situ and after iso-lation was studied recently.27,28 Follicles cryo-preserved in situ in 1.5 M DMSO and 3.0 M EGmaintained a high percentage of morphologi-cally normal follicles after thawing. For isolatedfollicles, DMSO and EG (both at 1.5 M), wereeffective. The objective of the present study wasto evaluate the in vitro survival of goat pre-antral follicles cryopreserved (in situ or iso-lated) using DMSO or EG as cryoprotectant.

MATERIALS AND METHODS

Chemicals

All chemicals and media used in this studywere purchased from the Sigma ChemicalCompany (St. Louis, MO). For all of the media,the pH was adjusted to 7.4.

Source of ovaries

This study was divided into two experiments.In each experiment, 10 ovaries from 5 adultmixed-breed goats were obtained at a local abat-toir. Ovaries were trimmed of adhering tissueand washed in 70% alcohol, and then in phos-phate-buffered saline (PBS) and transported tothe laboratory within 1 h in PBS at 20°C.

Experiment 1—Cryopreservation and in vitroculture of ovarian tissue

Ovarian tissue preparation and freezing/thawingprocedure: At the laboratory, the ovarian pairfrom each animal (five pairs) was divided intonine pieces of 3 � 3 � 1 mm. One ovarian frag-ment was taken randomly and immediatelysubmitted to follicular isolation and viabilityevaluation (control). Two fragments were usedfor in vitro culture (fresh control), and the re-maining six fragments were cryopreserved inDMSO or EG (Fig. 1).

For freezing, ovarian fragments were placedindividually in 2.0-mL vials and equilibrated in1.8 mL of minimum essential medium (MEM)supplemented with 10% fetal bovine serum(FBS), i.e., modified MEM (MEM�), with 1.5 MDMSO (n � 3) or 3.0 M EG (n � 3), accordingto previous observations (unpublished data).After an equilibration period (20 min), vialswere transferred to a programmable freezer(Freeze Control, CryoLogic Pty Ltd, Waverley,Australia) at 20°C. Vials were cooled at 2°C/min to �7°C and seeded manually by touchingthem with forceps prechilled in liquid nitrogen.The specimens were held at this temperaturefor 15 min and then cooled at 0.3°C/min to�30°C and thereafter at 0.15°C/min to �33°C.Then the specimens were plunged into liquidnitrogen (�196°C) and stored for up to 5 daysbefore thawing. The freezing program used inthis study was chosen according to proceduresdeveloped for ovarian tissue6–8,12,28–31 and iso-lated preantral follicles.10,11,26 For thawing,vials were taken from the liquid nitrogen,warmed at room temperature for 1 min, andimmersed in water at 37°C until the ice melted.For cryoprotectant removal, samples werewashed three times for 5 min in MEM� at roomtemperature, as described.28

In vitro culture of small preantral follicles insitu: Ovarian fragments (fresh, n � 2; cryopre-served using 1.5 M DMSO, n � 2; and 3.0 MEG, n � 2) were transferred to one well of a 24-well culture dish (Biosystems) containing 2 mLof culture medium. Tissues were cultured us-ing a modification of the method described byFigueiredo et al.32 for bovine preantral follicles:MEM supplemented with antibiotics (100�g/mL of penicillin, 100 �g/mL of streptomi-

CULTURE OF CRYOPRESERVED OVARIAN TISSUE 291

cyn), 0.25 �g/mL amphotericin B, 10% FBS, ITS(insulin 6.25 �g/mL, transferin 6.25 �g/mL,and selenium 6.25 ng/mL), 0.23,0 mM pyru-vate, 2 mM glutamine, and 2 mM hypoxan-thine. All reagents were obtained from SigmaChemical Co. (St. Louis, MO). Every 24 h, themedium was replaced with fresh medium.Ovarian fragments were cultured for 1 or 5days.

Experiment 2—Cryopreservation and in vitroculture of isolated preantral follicles

Isolation of small preantral follicles: Preantralfollicles were isolated from ovaries using a me-chanical procedure developed for caprine pre-antral follicles.33 Briefly, the ovarian tissue wascut into small fragments using a tissue chop-per (The Mickle Laboratory Engineering Co.,Gomshal, Surrey, UK) adjusted to 75 �m. Theovarian fragments were placed in MEM� andthen pipetted 40 times with a large Pasteurpipette (diameter, �1,600 �m) and 40 timeswith a smaller pipette (diameter, �600 �m).The suspension was filtered successively

through 500- and 100-�m nylon mesh filters. Ineach replicate, one aliquot of 100 �L contain-ing at least 50 follicles was analyzed immedi-ately after mechanical isolation (day 0), twoaliquots were used for in vitro culture (freshcontrol), and the remainder were cryopre-served in DMSO or EG (Fig. 2).

Freezing/thawing procedure: Vials (n � 6) con-taining follicular suspension were equilibratedin 1.5 M DMSO or EG for 20 min at 20°C, ac-cording to previous experiments.34 Then, sam-ples were frozen and thawed as describedabove for Experiment 1. For cryoprotectant re-moval, samples containing follicles were di-luted in 2.7 mL of MEM�, equilibrated for 5min at room temperature, and then centrifugedat 90 � g for 2 min. This procedure was per-formed a second time and follicles were finallysuspended in 0.9 mL of MEM�, modified fromJewgenow et al.26

In vitro culture of isolated preantral follicles: Forisolated preantral follicles, 0.5-mL freshaliquots (n � 2) and a follicle suspension cryo-preserved in 1.5 M DMSO (n � 2) or EG (n � 2) were transferred to 0.5 mL of culture

RODRIGUES ET AL.292

Ovarian pair

9 fra gments

3 fra gments 3 fra gments 3 fra gments

Cryopreservation (1.5 M DMS O)

Cryopreservation (3.0 M EG)

Follicular isolation

(Day 0 - control)

Culture (Day 1)

Culture (Day 5)

Viability assessment

Follicular isolation (Day 0)

Culture (Day 1)

Culture (Day 5)

Follicular isolation (Day 0)

Culture (Day 1)

Culture (Day 5)

Follicular isolation

Viability assessment

Follicular isolation

Viability assessment

Follicular isolation

Viability assessment

Viability assessment

Viability assessment

FIG. 1. Protocol design of experiment 1 for cryopreservation and in vitro culture of caprine ovarian tissue.

medium. Follicles were cultured as describedabove, except that the culture medium was notreplaced. Instead, 0.5 mL of medium wasadded at days 1 and 3 of culture.

Follicular growth and viability assessment: Af-ter follicular isolation, small preantral follicles(� 35 �m) were evaluated. Follicular diameterwas measured using an ocular micrometer be-fore and after in vitro culture. Follicular viabil-ity was evaluated immediately after isolation(control), and after 1 and 5 days of culture. A100-�L aliquot of follicular suspension wassupplemented with 5 �L of 0.4 % Trypan Bluesolution. After 1 min, the sample was viewedunder an inverted microscope and follicleswere classified as alive (not stained) or dead(stained).

Statistical analysis: All data from Experiments 1and 2 were analyzed using the SAS statistical soft-ware. The number of live follicles was examinedby the General Linear Model (GLM) procedure,and the independent variable used in the modelwas treatment. Differences among viability, anddiameter, of preantral follicles noncryopreservedor cryopreserved in situ (Experiment 1) or isolated(Experiment 2) on days 0, 1, and 5 of culture werechecked by a Duncan’s test. Values were consid-ered statistically significant when p � 0.05.

RESULTS

Table 1 shows the percentage of live follicleson days 0, 1, and 5 of in vitro culture, both fresh

CULTURE OF CRYOPRESERVED OVARIAN TISSUE 293

Ovarian pair

Follicular isolation – 9 aliquots (0.9 mL)

3 aliquots 3 aliquots 3 aliquots

Cryopreservation (1.5 M DMS O)

Cryopreservation (1.5 M EG)

Viability assessment (Day 0 - control)

Culture (Day 1)

Culture (Day 5)

Viability assessment

(Day 0)

Culture (Day 1)

Culture (Day 5)

Viability assess ment

(Day 0)

Culture (Day 1)

Culture (Day 5)

Viability assessment

Viability assessment

Viability assessment

TABLE 1. PERCENTAGE (MEAN � SD) OF LIVE PREANTRAL FOLLICLES ON DAYS 0, 1, AND 5 OF CULTURE, BOTH FRESH AND CRYOPRESERVED IN SITU

Live preantral follicles (%)

Treatment Day 0 Day 1 Day 5

Fresh (control) 84 � 6.9aA 81 � 10.6aA 68 � 10.7aA

Frozen in 1.5 M DMSO 91 � 3.3aA 64 � 5.3bAB 60 � 7.4bA0Frozen in 3.0 M EG 81 � 7.4aA 58 � 3.7bBA 45 � 4.4bA0

a,bValues within rows are different (p � 0.05).A,BValues within columns are different (p � 0.05).

FIG. 2. Protocol design of Experiment 2 for cryopreservation and in vitro culture of isolated caprine preantral folli-cles.

and cryopreserved in situ (Experiment 1). Infresh tissue, percentages of viable follicles ondays 0, 1, and 5 were similar. On the otherhand, follicles from tissue cryopreserved in 1.5M DMSO or 3.0 M EG had lower viability af-ter 1 and 5 days of culture when compared today 0. When compared within the same day (0,1, or 5) of culture, no difference was observedon the percentage of live follicles among treat-ments (fresh follicles and follicles cryopre-served in both cryoprotectants), except for 3.0M EG at day 1 of culture.

Table 2 shows the percentage of live follicleson days 0, 1, and 5 of in vitro culture, both freshand cryopreserved after isolation (Experiment2). The percentage of viable follicles was loweron days 1 and 5 of culture when compared today 0, both for fresh follicles and follicles cry-opreserved in 1.5 M EG, but not for follicles cry-opreserved in 1.5 M DMSO. On day 0, the per-centage of live follicles did not differ betweenfresh and cryopreserved follicles for both cryo-

protectants. On the other hand, on day 1 of cul-ture, the percentage of viable follicles waslower after cryopreservation in 1.5 M EG thanin 1.5 DMSO or fresh follicles. Moreover, onDay 5 the percentage of viable follicles previ-ously cryopreserved in EG was lower than thepercentage of viable fresh follicles. Alive anddead preantral follicles are shown in Fig. 3.

In the present study, the diameter of culturedfollicles, both fresh and after cryopreservation,did not differ statistically among treatments ordays of culture, both for follicles cultured in situ(Fig. 4) and after isolation (Fig. 5).

DISCUSSION

The present study showed that it is possibleto cryopreserve small caprine preantral folliclesisolated or in situ using DMSO. These folliclescan survive for 5 days in vitro, with 60% viablefollicles when cryopreserved and cultured in

RODRIGUES ET AL.294

TABLE 2. PERCENTAGE (MEAN � SD) OF LIVE PREANTRAL FOLLICLES ON DAYS 0, 1, AND 5 OF CULTURE, BOTH FRESH AND CRYOPRESERVED AFTER ISOLATION

% of live preantral follicles

Treatments Day 0 Day 1 Day 5

Fresh (control) 94 � 2.4aA 80 � 3.1bA 81 � 5.7bAB

Frozen in 1.5 M DMSO 85 � 5.4aA 74 � 3.6aA 77 � 2.5aAB

Frozen in 1.5 M EG 85 � 5.0aA 51 � 6.5bB 63 � 6.6bBB

a,bValues within rows are significantly different (p � 0.05).A,BValues within columns are significantly different (p � 0.05).

FIG. 3. Alive (A, not stained; bar represents 23 �m; 400�) and dead (B, stained by Trypan Blue; bar represents 40 �m; 400�) caprine preantral follicles.

situ and 77% viable follicles when isolated.Nevertheless, the viability of follicles cryopre-served in EG decreased rapidly after a shorttime in culture (1 day).

In Experiment 1, the percentage of live folli-cles in the control was similar before and afterculture for 1 and 5 days, whereas in cryopre-served tissue, these percentages decreased ondays 1 and 5 compared to day 0. Maintenanceof follicular viability could be due to a bettercontact with the components of ovarian extra-cellular matrix, which was absent or reducedin cryopreserved follicles. Zhao et al.35 haddemonstrated that in murine preantral folliclescultured in vitro, the best growth and survivalrates were obtained when interfollicular inter-actions were maintained. The extracellular ma-trix can also be an important element, once itprovides a favorable condition for differentia-tion36 and certainly for cellular survival. On thecontrary, in Experiment 2, the viability of small

unfrozen preantral follicles was significantlylower after 1 and 5 days of culture comparedto day 0 (zero). We suggest that the reduced vi-ability can be due to an inefficient interactionbetween follicles, as well as the absence of ex-tracellular matrix.

In Experiments 1 and 2, viability of cryopre-served follicles was reduced after culture.Therefore, cryopreservation affected follicularviability in culture. Newton et al.16 reportedthat after 5 days of culture, about 60% of ovinepreantral follicles from cryopreserved ovariantissue extruded their oocytes or became mis-shapen. According to these authors, folliculardamage may not be observed after short-termculture.16 On the other hand, after prolongedculture, the basement membrane may be dam-aged. Previous studies37,38 reported that main-taining follicular viability is the major problemduring culture of preantral follicles. Figueiredoet al.31 observed that the viability of bovine pre-

CULTURE OF CRYOPRESERVED OVARIAN TISSUE 295

FIG. 4. Diameter of fresh (control) and cryopreserved in situ preantral follicles on days 0, 1, and 5 of in vitro cultureof ovarian tissue.

FIG. 5. Diameter of fresh (control) and cryopreserved isolated preantral follicles on days 0, 1, and 5 of in vitroculture.

antral follicles was decreased by many factors,including excessive manipulation before cul-ture, time expended between isolation and cul-ture, and culture medium composition. In ourstudy, cryopreserved follicles were more sus-ceptible, because viability decreased even aftera short period of culture (1 day). Paynter et al.30

showed that the percentage of morphologicallynormal follicles progressively decreased after4, 24, and 48 h of culture. According to theseauthors, oxygen, nutrients, and diffusion of au-tocrine factors may be a limiting factor for sur-vivability of preantral follicles cultured withinovarian tissue. We believe that these events aremore critical after damage usually observed inovarian tissue during the cryopreservationprocess.

Within the same day of culture, except forday 1, there were no differences between freshand cryopreserved follicles in situ. Accordingto Newton et al.,39 the percentage of morpho-logically normal human follicles in ovarian tis-sue cryopreserved in DMSO (74%) or EG (84%)did not differ from that observed in fresh ovar-ian tissue (100%) after transplantation intomice. According to Oktay et al.,2 small preantralfollicles are extremely resistant and their via-bility is not affected when they are cryopre-served. One of the factors that determines thisresistance to freezing is the small size of thesefollicles, which allows the cryoprotectant topermeate the cells rapidly. Moreover, the rela-tively quiescent and undifferentiated stage ofthe oocyte is also important, reducing damageto organelles.2

No difference in the viability of isolated fol-licles was observed between fresh and cryo-preserved follicles on day 0. However, on day1 the viability of follicles cryopreserved usingEG was lower than fresh follicles and folliclescryopreserved in DMSO, and on day 5 the vi-ability of follicles cryopreserved using EG waslower than fresh follicles, but not lower thanfollicles cryopreserved using DMSO. In workwith human ovarian tissue, Hovatta et al.8showed that when morphological analysis isperformed immediately after freezing/thaw-ing only small damages are revealed, whereasmore serious injuries can be observed when aperiod of incubation follows the cryopreserva-tion. In mice, Cortvrindt et al.10 also showed

that the in vitro growth and maturation rateswere similar between fresh follicles and thosecryopreserved in 1.5 M DMSO. The results ofthe present work, as well as the report ofCortvrindt et al.,10 demonstrated that DMSO isa good cryoprotectant for small preantral folli-cles, perhaps due to its great permeability.40

Furthermore, our results showed that follicularviability after 5 days of culture was similar be-tween cryopreservation using DMSO and EG,both in situ and after isolation. These resultsdiffer from those described by Amorim,41 whoshowed that the viability of ovine primordialfollicles after 4 days in culture was significantlyhigher when cryopreserved in EG than inDMSO. This may be due to differences betweenspecies, as well as different protocols used forcryopreservation and culture. Many studieshave shown that both DMSO8,29,42 and EG29,39

can be used successfully to cryopreserve pre-antral follicles.

In the present study, follicular diameter didnot change significantly during in vitro culture,neither for fresh or cryopreserved follicles, inboth experiments. Similarly, Sirard and Co-enen43 cultured bovine preantral follicles for�3 months and, although follicular viabilitywas maintained, follicular growth was not ob-served.

In conclusion, caprine preantral follicles canbe successfully cryopreserved in situ, keepingfollicular viability similar between fresh andcryopreserved follicles after 1 and 5 days of cul-ture.

ACKNOWLEDGMENTS

This work was supported by CNPq. AnaPaula R. Rodrigues is a recipient of a grant fromFUNCAP Brazil. The authors thank Dr. Edil-berto Silveira, Ph.D. (CENAUREMN), andAKZO-NOBEL for logistical support, and Z.R.Madeira for reviewing the manuscript.

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Address reprint requests to:Dr. Ana Paula R. Rodrigues

Universidade Estadual do CearáFaculdade de Veterinária

Programa de Pós-graduação em Ciências VeterináriasAv. Paranjana, 1700

Campus do Itaperi CEP 60740-000 Fortaleza,Ceará, Brasil

E-mail: aprrodrigues@yahoo.com.br

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