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MOLECULAR REPRODUCTION AND DEVELOPMENT 71:389397 (2005)

Developmental Capability of Denuded Bovine

Oocyte in a Co-Culture System With IntactCumulus-Oocyte Complexes: Role of Cumulus Cells,Cyclic Adenosine 30,50-Monophosphate,and Glutathione

ALBERTO M. LUCIANO,* VALENTINA LODDE, MATTEO S. BERETTA, SILVIA COLLEONI,

ANTONIO LAURIA, AND SILVIA MODINA

Institute of Anatomy of Domestic Animals, Histology and Embryology, Faculty of Veterinary Medicine,University of Milan, Italy

ABSTRACT Cumulus oophorus cells havebeen implicated in the regulation of female gametedevelopment, meiotic maturation, and oocyte-sperm

interaction. Nevertheless, the specific role of cumuluscells (CCs) during the final stages of oocyte maturation

and fertilization processes still remains unclear.Several studies have been conducted in order to clarifythe role of follicular cells using culture systems where

denuded oocytes (DOs) were co-cultured with isolated

CCs, or in the presence of conditioned medium.However, those attempts were ineffective and the

initial oocyte competence to become a blastocystafter fertilization was only partially restored. Aim of

the present study was to analyze the effect of the

interactions between somatic cells and the femalegamete on denuded oocyte developmental capabilityusing a system of culture where CCs were present asdispersed CCs or as intact cumulus-oocyte complexes

(COCs) in co-culture with oocytes freed of CC invest-

ment immediately after isolation from the ovary.Moreover, we analyzed the specific role of cyclic

adenosine 3050 monophosphate (cAMP) and glu-tathione (GSH) during FSH-stimulated maturation ofdenuded oocyte co-cultured with intact COCs. Our data

confirm that denuded oocyte has a scarce develop-

mental capability, and the presence of dispersed CCsduring in vitro maturation (IVM) does not improve their

developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVMpartially restores their developmental capability. The

absence of CCs investment causes a drop of cAMP

content in DOs at the beginning of IVM and the additionof a cAMP analog in the culture medium does notrestore the initial oocyte developmental competence.

The relative GSH content of denuded oocyte matured inpresence of intact COCs is consistent with the partial

recovery of their developmental capability. However,the complete restoration of a full embryonic develop-

mental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro

fertilization (IVF). Our results suggest that the direct

interaction between oocyte and CCs is not essentialduring IVM and IVF of denuded oocyte. We hypothe-size that putative diffusible factor(s), produced by CCs

and/or by the crosstalk between oocyte and CCs in theintact complex, could play a key role in the acquisition

of developmental competence of the denuded femalegamete. Mol. Reprod. Dev. 71: 389397, 2005. 2005 Wiley-Liss, Inc.

Key Words: denuded oocyte; cumulus-oocyte com-plex; cAMP; cysteamine; glutathione; secreted factors

INTRODUCTION

In the antral ovarian follicle, the cumulus oophorus isa group of closely associated granulosa cells (namelycumulus cells (CCs)), which plays a crucial role duringoocyte growth and development, and ultimately duringthe maturation and fertilization processes in mamma-lian oocytes [reviewed in Tanghe et al. (2002)].

CCs exert important biological function: before ovula-tion they support oocyte cytoplasmic maturation andshortly after ovulation participate in the complexmechanisms controlling sperm oocyte interaction(Tesarik, 1990). CCs communicate with each other andwith the oocyte by means of an extensive network ofgap junctions (Gilula et al., 1978), which permits the

two-way transfer of small molecules such as nutrientsand messengers between somatic and germ cells (Moor

2005 WILEY-LISS, INC.

Grant sponsor: PRIN; Grant number: 12-2-5159205-12; Grant sponsor:FIRST; Grant number: 12-2-5159205-42.

Silvia Colleonis present address is Laboratorio di Tecnologie dellaRiproduzione, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy.

*Correspondence to: Alberto M. Luciano, Istituto di Anatomia degliAnimali Domestici con Istologia ed Embriologia, Facolta di MedicinaVeterinaria, Universita degli Studi di Milano, Via Celoria, 10 20133Milano, Italy. E-mail: alberto.luciano@unimi.it

Received 15 December 2004; Accepted 4 February 2005Published online 1 April 2005 in Wiley InterScience

(www.interscience.wiley.com).DOI 10.1002/mrd.20304

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et al., 1980; Dekel etal.,1981; Eppig, 1982; deLooset al.,1991). This coupling is essential for supplying oocytewith metabolites like pyruvate and glutathione (GSH)that are produced by the accompanying CCs (Tangheet al., 2002). GSH is the major nonprotein sulfhydrylcompound in mammalian cells and is well known to

play an important role in protecting cells against thedestructive effects of reactive oxygen intermediates andfree radicals (Meister, 1983). Moreover, GSH partici-pates in sperm decondensation and male pronucleusformation, during fertilization in hamster (Perreaultet al., 1988), pig (Yoshida et al., 1993), and cow(Sutovsky and Schatten, 1997). The addition of GSHsynthesis precursors, such as cysteine or cysteamine, tothe culture medium improved the maturation efficiencyby increasing the oocyte GSH content in mouse (deMatos et al., 2003), cow(de Matos et al., 1997), sheep (deMatos et al., 2002), and buffalo (Gasparrini et al., 2003).CCs play an important role in GSH synthesis because

the increase of oocyte GSH level is dependent by thepresence of cumulus mass while denuded oocytes (DOs)had only a limited capability to synthesize GSH (deMatos et al., 1997).

The direct transfer of substances between CCs andoocyte is also responsible for the maintenance of meioticarrest (Buccione et al., 1990). Gonadotropins FSHand LH exert their function by activating membranereceptors of target cells, which, in turn, activateadenylate cyclase leading to the production of cyclicadenosine 3050 monophosphate (cAMP). In particular,cAMP, which is the second messenger for signaltransduction pathway of gonadotropins, seems to playa pivotal role both in keeping the oocyte arrested in

prophase I and in inducing meiotic resumption. cAMPcan move between somatic andgerminalcompartments,and relative changes in cAMP concentration, modulatedby specific phosphodiesterase, can account for the re-sponse of the oocyte to gonadotropin stimulation (Contiet al., 1998). Compounds that elevate intracellularcAMP can inhibit or delay the resumption of meiosis,whereas a transient elevation of cAMP has been de-monstrated to induce oocyte maturation (Dekel et al.,1988; Downs et al., 1988; Luciano et al., 1999, 2004).

Besides the direct interaction between oocyte andCCs, there is some evidence that factors secreted byCCs are very important in promoting oocyte maturation

and acquisition of developmental competence. Duringnatural gonadotropin-induced ovulation at mid-cycle,the meiotic resumption of the oocyte is accompaniedwith the expansion of cumulus oophorus cells, which isinduced in vitro by FSH (Salustri et al., 1990b) via amechanism that appears to be mediated by cAMP in ratand cow (Dekel and Kraicer, 1978; Ball et al., 1983).Expanded cumulus matrix is composed of glycosamino-glycans such as hyaluronic acid (Ball et al., 1982), whichare secreted by CCs and that may contribute to themicroenvironment essential to sperm capacitation andfertilization (Tanghe et al., 2003).

In bovine species, removal of CCs at the beginning ofin vitro maturation (IVM) or shortly before in vitro

fertilization (IVF) (Fukui and Sakuma, 1980; Zhanget al., 1995; Fatehi et al., 2002) has been shown to bedetrimental to the correct oocyte maturation andfertilization.

Several studies have been conductedin order to clarifythe specific role of follicular cells in the maturation and

fertilization process using culture systems where DOswere co-cultured with isolated CCs, either in suspensionor monolayer, or in the presence of conditioned medium(Hashimoto et al., 1998; Ikeda et al., 2000; Tanghe et al.,2003). However, those attempts were ineffective and theinitial oocyte developmental competence to undergofertilization and blastocyst formation was only limitedlyrestored (Geshi et al., 2000).

Aim of the present study was to analyze the interac-tions between somatic cells andthe female gamete usinga system of culture where CCs were added as intactCOCs in co-culture with oocytes freed of cumulusinvestment prior to the beginning of IVM.

In particular, we analyzed the specific role of cAMPand GSH intra-oocyte contents during denuded oocyteFSH-stimulated maturation on their subsequent devel-opmental capability.

MATERIALS AND METHODS

Chemicals and Reagents

All chemicals and reagents were purchased fromSigma Chemical Company (St. Louis, MO), unlessotherwise stated.

Oocyte Collection, Selection,and In Vitro Maturation

Bovineovaries were obtained from a local abattoir andtransported to the laboratory, within 2 hr, in sterilesaline (9 g NaCl/L) maintained at 32 348C. As pre-viously described, only ovaries with more than 10follicles were processed for oocyte aspiration (Gandolfiet al., 1997). All subsequent procedures were performedat 36388C.

COCs were retrieved from 26 mm follicles with a 16-gauge needle mounted on an aspiration pump (COOK-IVF, Australia) with a vacuum pressure of 28 mm/Hg.The COCs were examined under a stereomicroscope,and only those medium-brown in color, with five or morecomplete layers of CCs and a finely granulated homo-

genous ooplasm were used. Selected COCs were thenwashed two times in M199 supplemented with HEPES20 mM and 0.4% of bovine serum albumin (HM199) andtwo times in the maturation medium, according to theexperimental design. The whole procedure was per-formed in approximately 30 min.

In accord to the experimental design, oocytes werecultured in basic maturation medium (bMM) that wasTCM-199 supplemented with 0.68 mM L-glutamine,25 mM NaHCO3, 0.4% of bovine serum albumin, fattyacid free, and 0.2 mM of sodium pyruvate or completematuration media (cMM) that was bMM with 0.1 IU/mlof human recombinant FSH (rhFSH, Gonal-F, Serono,Rome, Italy). Oocytes were matured in 500ml of medium,

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in four-well dishes (NUNC, VWR International, Milan,Italy), andincubated for 24 hr at 38.58C under 5%CO2 inhumidifiedair. Groupsof about 30 oocytes were culturedeither as intact COCs or DOs, obtained by mechanicalremoving of CCs from COCs by the use of vortex (2 min,35 Hz) after the selection procedure.

According to the experimental design, groups of DOswere co-cultured either in presence of intact COCs ordispersedCCs. For theformer, about 15 COCs andabout15 DOs were co-cultured in a 1:1 ratio during IVM(Fig. 1). For the latter, about 30 DOs were cultured inthe presence of dispersed CCs obtained by mechanicalremoval from 15 complexes, as above described. DOs co-cultured in the presence of intact COCs during IVM areindicated as coDOs while those cultured in presence ofCC are indicated as DOsCC.

In Vitro Fertilization

After maturation, oocytes were fertilized as pre-

viously described (Luciano et al., 1999). Briefly, thecontent of a straw of cryopreserved bull spermatozoa(CIZ, S. Miniato Pisa, Italy) was thawed and cellsseparated on a 4590% Percoll gradient. Sperms werecounted and diluted to a final concentration of 0.5 106

spermatozoa/ml in fertilization medium that wasTALP supplemented with 0.6% (w/v) BSA fatty acidfree, 10 mg/ml heparin, 20 mM penicillamine, 1 mMepinephrine, and 100 mM hypotaurine. Groups of about30 COCs or DOs or coDOs (Fig. 1A), were incubated in300 ml of fertilization medium for 18 hr in four-welldishes at 38.58C under 5%CO2 in humidified air. BeforeIVF, DOs matured in presence of intact COCs (coDOs)were separated from COCs by using a narrow-bore

pipette.According to the experimental design, groups of about

30 coDOs were maintained in co-culture also duringIVF. This group is indicated as coDOF.

Embryo Culture

Embryo culture medium was a modified syntheticoviduct fluid (SOF (Tervit et al., 1972)), buffered with

25 mM of NaHCO3, supplemented with MEM essentialand nonessential aminoacids, 0.72 mM of sodiumpyruvate, 2.74 mM of myo-inositol, 0.34 mM of sodiumcitrate, and with 5% of calf serum (SOF-C). Embryomanipulation medium was SOF supplemented with0.3% (w/v) BSA fraction V, fatty acid free, MEM

essential and nonessential aminoacids, 0.72 mM ofsodium pyruvate, and buffered with 10 mM of HEPESand 5 mM of NaHCO3 (SOF-R).

At the end of fertilization, presumptive zygotes werevortexed for 2 min in 500 ml of SOF-R, rinsed two times,and then transferred in groups of about 30 in 400 ml ofSOF-C under 400 ml of mineral oil. After IVF, coDOFswere separated from intact COCs by using a narrow-bore pipette before vortexing.

Incubation was performed in a culture box thatwas a small desiccators cabinet (Sigma-Aldrich cat#Z11,9059) with an internal volume of 4 L, with arubber gasket door for tight seal. The culture box was

modified with inlet and outlet gas tubing and placed in aconventional 5% CO2 incubator maintained at 38.58C.The box was supplied for 30 min with humified gasmixture composed by 5% CO2, 5 % O2,and90%N2 with aflux of 15 L/hr. After 48 hr post insemination, thenumber of uncleaved embryos was rapidly recorded andthe 4-well plate was placed back in the culture box. Thebox was supplied for 30 min with humified gas mixtureas above indicated. At the end of culture period (186 hrpost insemination), blastocyst rate was assessed andembryos were fixed in 60% methanol in PBS. Cell nucleiwere counted under fluorescence microscopy afterstaining with 0.5 mg/ml of propidium iodide.

Determination of IntracellularConcentration of cAMP in Oocyte

Intra-oocyte cAMP content was determined beforematuration, and after 3 hr of culture either in COCs,DOs, or coDOs.

COCs were denuded by vortex in SOF-R in which wasincluded 0.5mM of IBMX in order to preserve the oocytecAMP concentration (Luciano et al., 2004).The completeremoval of CCs from oocytes was monitored using astereomicroscope. DOs and coDOs were washed twotimes in the same buffer containing IBMX. Finally,oocytes were washed two times in fresh SOF-R in theabsence of IBMX, transferred in a minimum volume of

washing buffer ranging between 3 and 5 ml i n a neppendorf tube, snap frozen in liquid nitrogen andstored at 208C until assayed. Intracellular concentra-tion of cAMP was determined by competitive enzymeimmunoassay system (EIA, Biotrak, Amersham LifeScience, Milan, Italy) with acetylation protocol forhighest test sensitivity, according to the procedureprovided with the kit. At the end of the procedures,optical density of samples was determined in a platereader at 450 nm within 30 min. Each sample wastested in triplicate in each experiment. The assay wasvalidated by adding increasing numbers of oocytes (20,50, and 100) to a constant volume of extraction mediumand recovering a proportional amount of cAMP.

Fig. 1. Co-culture system of denuded oocytes in presence of intactCOCs during IVM.

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Determination of Intra-oocyte GSHConcentration

After isolation, oocytes were matured in cMM, inpresence or absence of cysteamine, either as COCs, Dosand coDOs. At the end of IVM, COCs were stripped freeof CCs and washed three times in PBS containing 1 mg/ml of PVA. Groups of 5 15 matured oocytes weretransferred under a stereomicroscope in the bottom ofan eppendorf tube with a narrow-bore pipette and thebuffer was successively removed by aspiration. Sampleswere snap frozenand stored at808C untilassayed. Theoocyte GSHcontent was determined by 5,50-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recyclingmicro-GSH assay according to Baker et al. (1990) andP.J. Hansen, University of Florida, (online protocol:http://www.animal.ufl.edu/hansen) with slight modifi-cations. Briefly, oocytes were thawed on melting ice and50 ml of deionized water were added to each sample.Each microtube was then frozen in liquid nitrogen and

successively thawed. This procedure was repeated threetimes. Standards containing from 0.19 to 200 pmol in50 ml were prepared in water, simultaneously with thesamples. The samples and standards were kept on iceuntil being loaded in the microtiter plate. A volume of50 ml of each sample and standards was added in a 96-well microtiter plate. Reaction mixture was freshlyprepared with 0.15 mM of DTNB, 0.2 mM of NADPH,and 1.0 U/ml GSH reductase (final concentrations) in0.1 M phosphate buffer, pH 7.8, supplemented with1 mM of EDTA. Immediately,0.1 ml waspipetted in eachwell andthe platewas analyzed at 405nm in a microtiterplate reader (SpectraCount, Packard, USA) with oneinitial mixing and repeated-reads functions at 2 minintervals for 30 min.

Experimental Design

Experiment 1: Effect of co-culture of DOs withintact COCs or dispersed CCs during FSH-stimu-lated IVM on the subsequent embryonic develop-mental competence. This experiment was aimed toassess the effect of CCs either as intact COCs ordispersed CCs in co-culture during FSH-stimulatedIVM of DOs on their subsequent developmental cap-ability. Part of selected COCs was denuded by vortex.Groups of DOs were maturedeither as DOs, coDOs, or in

presence of dispersed CCs, either in bMM or cMM.Groups of intact COCs were matured in the sameconditions, as control.

After maturation, oocytes were fertilized and pre-sumptive zygotes were cultured as above described.

Experiment 2: Role of cAMP during denudedoocyte maturation. In the first part of the experi-ment, we analyzed the effect of co-culture of denudedoocyte on the cAMP content during 3 hr of IVM. Groupsof DOs, coDOs, and intact COCs were cultured in cMM.

A group of DOs was cultured also in bMM, in absence ofhormonal stimulation, as control. After 3 hr of matura-tion, oocyte cAMP content in experimental groups wasevaluated as above described.

Since FSH stimulation apparently did not induce anappropriate intra-oocyte cAMP concentration, in thesecond part of the experiment, we evaluated the effect ofcAMP analog administration during IVM of co-cultureddenuded oocyte. For this purpose, groups of coDOs werecultured in cMM supplemented with non-inhibitory

concentration 0.1, 0.05, and 0.01 mM of dybutyril-cAMP(dbcAMP) (Homa, 1988). After maturation, coDOs wereseparated from intact complexes then fertilized andpresumptive zygotes were cultured as above described.

Experiment 3: Role of GSH during denudedoocyte maturation. In the first part of the experi-ment, we analyzed the effect of the co-culture systemand cysteamine administration on the intra-oocytecontent of GSHat the endof maturation process. Groupsof DOs, coDOs, and intact COCs were cultured in cMMin presence or absence of 100 mM of cysteamine. After24 hr of maturation, oocyte GSH content was evaluatedas above indicated.

In the second part of the experiment, we analyzed theeffect of cysteamine administration during IVM ofdenuded oocyte, incubated either alone or in co-culturewith intact COCs, on the subsequent embryonic devel-opment. Groups of DOs, coDOs, and intact COCs werematured in cMM in presence or absence of 100 mM ofcysteamine. After maturation, oocytes were fertilizedand presumptive zygotes were cultured. Cleavage andblastocyst rates were assessed as above described.

Experiment 4: Effect of co-culture during bothIVM and IVF on embryonic development ofDOs. In order to analyze the effect of co-culture duringboth maturation and fertilization processes on thedevelopmental capabilities of denuded oocyte, groups

of oocytes were matured as coDOs in cMM. Aftermaturation, groups of coDOs were kept as coDOFs.

After IVF, coDOFs were separated from intact com-plexes then presumptive zygotes were cultured and em-bryonic development was assessed as above described.

Statistical Analysis

All experiments were replicated five times. Resultsare expressed as meanSEM. Data were analyzedusing one-way ANOVA followed by Fishers protectedleast significant difference test (SuperANOVA, AbacusConcepts, CA). Probabilities of less than 0.05 wereconsidered statistically significant.

RESULTS

Experiment 1: Effect of Co-Culture of DOsWith Intact COCs or Dispersed CCs During

FSH-Stimulated IVM, on EmbryoDevelopmental Competence

As reported in Table 1, the removal of CCs before IVMsignificantly decreased the number of oocytes thatcleaved after 48 hr post insemination and the numberof those that reached the blastocyst stage after 186 hrpost insemination. The administration of FSH duringIVM had no effect on the cleavage and blastocyst rates ofDOs. The presence of dispersed CCs in the maturation

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environment did not improve the developmental cap-ability of DOsCCs and their cleavage and blastocystrates were similar to those of DOs. On the contrary, thepresence of intact COCs during FSH-stimulated IVM,

partially restored the developmental capability of DOs.Cleavage rate of coDOs was similar to that of intactCOCs while blastocyst rate was higher than DOs(P< 0.001) but significantly lower than intact COCs.

The presence of the gonadotropin during oocytematuration of intact COCs induced a cleavage andblastocyst rate of 94.1 and34.8, respectively, while in itsabsence, intact COCs exhibited a developmental poten-tial similar to that of DOs.

Experiment 2: Role of cAMP During DenudedOocyte Maturation

The intra-oocyte content of cAMP significantlyincreased in all groups after 3 hr of IVM, compared to

time 0 hr (Table 2). However, cAMP was significantlyhigher in oocytes cultured as intact COCs than denudedoocyte, either cultured alone (DOs), or in co-culture(coDOs).

The administration of dbcAMP during IVM ofdenuded oocyte co-cultured with intact COCs did notincrease the percentage of embryos that reached theblastocyst stage of development at the end of culture(Table 3). All the concentrations of dbcAMP added tothe culture media in fact resulted in a cleavage andblastocyst rate similar to coDOs matured in cMM alone,significantly lower than intact COCs.

Experiment 3: Role of GSH During DenudedOocyte Maturation

Before maturation, the average of intra-oocyte GSHcontent was 5.71 pmol/oocyte (Table 4). The absence of

CCs significantly reduced the GSH content in DOs after24 hr of IVM. However, the presence of intact COCs inco-culture maintained the GSH content of coDOs signi-ficantly higher than DOs. The administration of 100mMof cysteamine in the maturation medium induced asignificant increase of intracellular GSH concentrationbothin DOs and coDOs. At the end ofmaturation, coDOsshowed an oocyte GSH content similar to that retainedbefore the beginning of maturation.

Intact COCs showed a significant increase of GSHcontent after culture and the addition of cysteamineinduced the highest increase of intra-oocyte GSH levelafter 24 hr of IVM.

As reported in Table 5, the administration of cystea-mine during IVM of DOs induced a significant increaseof both the cleavage and the blastocyst rates, whichresulted similar to those obtained in coDOs, but did notfurther improve the developmental capability of coDOs.No differences were observed between COCs matured incMM and those treated with the GSH precursor duringIVM. Finally, no differences were observed in theblastocyst cell number among all treatments.

Experiment 4: Effect of Co-Culture DuringBoth IVM and IVF on Embryonic

Development of DOs

The presence of intact COCs since the beginning of

IVM and up to the end of IVF significantly improved thedevelopmental capability of DOs. As shown in Table 6,the percentages of cleaved embryos and blastocystsobtained from coDOFs were significantly higher thanthose from DOs co-cultured with intact COCs onlyduring IVM (coDOs), and similar to the percentagesobtained in the control group (COCs).

No differences were observed in the number of nucleiper blastocyst among all treatments.

DISCUSSION

The results of the present study provide evidencethat the addition of intact COCs, during both FSH-

TABLE 2. Effect of Co-Culture of DOs With IntactCOCs and rhFSH Administration During IVM onIntra-Oocyte cAMP Content After 3hr of Culture*

TreatmentIVM

mediumPresence of

FSHOocyte cAMP

content

Before culture 0.290.014a

DOs bMM 0.3610.042b

DOs cMM 0.3920.021b

coDOs cMM 0.3850.043b

COCs cMM 0.4970.035c

Different superscripts (a c) indicate statistical differences(P

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stimulated IVM and IVF of DOs, can completely restoretheir developmental capability (Table 6).

Similar to previous studies (Hashimoto et al., 1998;Geshi et al., 2000; Ikeda et al., 2000), our data show thata limited proportion of DOs are able to reach theblastocyst stage after fertilization (Table 1).

Previous works showed that the use of dispersedcumulus or granulosa cells co-culture, or conditionedmedia during IVM and/or IVF, is not able to restore thedevelopmental capability of DOs (Hashimoto et al.,1998; Ikeda et al., 2000; Tanghe et al., 2003). Accordingto the above studies, our results further confirm that thepresence of dispersed CCs in the maturation environ-ment does not improve the developmental capability ofoocytes deprived of their CCs investment.

We hypothesize that the results obtained in oursystem could be due to the preservation of the physio-logical interaction between the oocytes and CCs in theintact complex, which can be more beneficial to thedenuded oocyte. In fact, we have previously demon-

strated that disaggregated cumulus or granulosa cellsundergo rapidly to apoptosis during in vitro culture(Luciano et al., 2000).

Our results confirm that FSH does not affect thedevelopmental capability of DOs when cultured alone(Sirard et al., 1988). FSH exerts its function by activat-ing membrane receptors of target cells leading to theproduction of cAMP, by adenylate cyclase. cAMP acts asthe intracellular messenger for gonadotropins stimula-tion (Steele and Leung, 1993; Ji et al., 1996). The deter-mination of intra-oocyte cAMP concentration indicated

that the presence of FSH in the maturation media doesnot influence the cAMP concentration in DOs. On theotherhand,oocytesremovedfromintactCOCs,after3hrof culture in presence of FSH, showed the highestincrease of cAMP concentration as previously observed(Luciano et al., 2004). The localization of FSH receptor

in mammalian oocyte is still under investigation. Insome species like human,mouse, andpig (Patsoula et al.,2001, 2003; Meduri et al., 2002), the presence of FSHreceptor and/or its transcript has been demonstratedwhile in bovine species contrasting data have beenobtained. Our results support previous studies where inbovine antral follicles, FSH receptors were detected ongranulosa and CCs but not on the plasma membrane ofthe oocyte (van Tol et al., 1996). Furthermore, thissupports recentfindingsdemonstrating that an increasein cAMP, induced by FSH in ovarian somatic cells, isultimately communicated from CCs to theoocyte viagap

junctions (Webb et al., 2002).After 3 hr of culture, an increase of cAMP concentra-

tion was detected in DOs, significantly higher than thatretained by the oocyte at the beginning of culture. Theincrease of intra-oocyte cAMP was similar either inpresence or in absence of FSH but it was significantlylower than in oocytes cultured as intact COCs. Thisobservation could be explained by the fact that bovine aswell as mouse oocyte possesses itsown adenylate cyclase(Kuyt et al., 1988; Horner et al., 2003) and phospho-diesterase system (Tsafriri et al., 1996; Thomas et al.,2002) being able to modulate autonomously the intra-cellular cAMP content (Thomas et al., 2002). While highlevels of cAMP have been proposed as the regulatorymechanism to maintain the oocyte in meiotic arrest

(Homa, 1988; Bilodeau et al., 1993), a moderate increaseof intra-oocyte cAMP concentration has been suggestedas regulatory signal for meiotic resumption (Tsafririet al., 1972, 1996; Luciano et al., 2004). However, in ourculture system, the low developmental capability ofdenuded oocyte was not due to the absence of an ad-equate cAMP induction. In fact, none of the noninhibi-tory dbcAMP concentrations added to the culture mediashowed a restoring effect of the developmental compe-tence in DOs.

In our co-culture system, intracellular GSH concen-tration seems to play a significant role in the acquisitionof developmental competence of DOs. At the end of IVM,DOs showed a decrease of GSH level. However, when

TABLE 3. Effect of dbcAMP Administration During IVM on EmbryonicDevelopmental Capability of Denuded Oocytes*

Treatment Total oocytes % Cleaved% Blastocysts

on cleaved% Blastocysts

on total

coDOs 132 94.21.7 24.72.6b 23.42.7b

coDOs

0.1 dbcAMP 126 95.2

3.8 25.1

3.3

b

23.6

2.4

b

coDOs0.05 dbcAMP 129 92.63.7 20.25.0b 18.33.7b

coDOs0.01 dbcAMP 127 93.60.4 21.83.1b 20.53.0b

COCs 135 92.91.1 40.34.3a 37.43.8a

Different superscripts (a, b) within columns indicate statistical differences (P

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cultured in presence of cysteamine, they were able toretain an intra-oocyte GSH content significantly higherthan DOs. The ability of synthesizing GSH from itsprecursor cysteamine has been previously demon-

strated both in cumulus enclosed oocyte and denudedoocyte in bovine (de Matos et al., 1997) and porcine(Yamauchi and Nagai, 1999). A positive effect of GSHcontent on oocyte developmental competence has beendemonstrated in several mammalian species (de Matoset al., 1995, 2002, 2003; Gasparrini et al., 2003). TheGSHaccumulation during IVMof DOsand coDOs in ourexperiment could be related to their capability to reachthe blastocyst stage of development after fertilization.

While DOs cultured alone showed the lowest blastocystrate, the presence of cysteamine or intact COCs duringIVM of DOs induced a significant increase of develop-mental rate. In fact, oocyte GSH content and blastocystrate of DOs matured in presence of cysteamine and DOs

co-cultured with intact COCs (coDOs) were similar.The presence of intact COCs during IVM was able to

maintain the GSH concentration in coDOs significantlyhigher than in DOs matured alone and similar to theGSH content of DOs matured in presence of cysteamine.In fact, it could be hypothesized that the achievementof a minimal GSH concentration could promote animprovement of the developmental competence ofDOs.

Over the oocyte ability to synthesize GSH fromcysteamine precursor even in absence of CCs (de Matoset al., 1997), a direct role of intact COCs inthe co-cultureenvironment, in preventing a depletion of GSH storage

of the denuded oocyte, cannot be ruled out.However, the presence of COCs or the addition ofcysteamine during IVM of denuded oocytes can recover

only partially their initial developmental competence.Only the addition of intact COCs during both IVM andIVF promoted the complete restoration of the initialdevelopmental competence of DOs.

It has been previously demonstrated that rat granu-losa cells respond to gonadotropin stimulation byproviding a factor(s) that regulate the rate of oocytematuration and promotes the oocytes capability toundergo fertilization (Vanderhyden and Armstrong,1990). Moreover, Biskov et al. demonstrated that CCsof mouse COCs secrete a meiosis activating substancewhen stimulated with FSH (Byskov et al., 1997). Thisheat-stable substance diffuses into the oocyte and in aparacrine way activates the resumption of meiosis notonly in the whole complex but also in DOs. This couldsuggest that the intimate contact between the CCs andthe oocyte is not essential for resumption of meiosis butthis contact is needed for the initial production of the

meiosis activating substance.The complex of molecules, which constitute the ex-

tracellular matrixof CCs produced through thecumulusexpansion or mucification, mediates the achievement ofa full oocyte developmental capability during matura-tion and fertilization (Zhuo and Kimata, 2001). Duringgonadotropin-induced ovulation at mid-cycle, the num-ber of gap junction decreases in parallel with the meioticresumption of the oocyte (Eppig, 1982; Larsen et al.,1987), and this event is accompanied by the progressiveCCs expansion and hyaluronic acid synthesis. Bothprocesses are induced in vitro by FSH (Salustri et al.,1990a) via a mechanism that appears to be mediated by

cAMP in rat (Dekel and Kraicer, 1978) and cow (Ballet al., 1983). Mucification in vitro is consistent withhyaluronic acid synthesis starting at 3 hr from the

TABLE 5. Effect of the Administration of Cysteamine 100 mM During IVM on Developmental Competence ofDenuded Oocyte Matured in the Presence of Absence of Intact COC*

TreatmentIVM

cysteamineTotal

oocytes % Cleaved% Blastocysts

on cleaved% Blastocysts

on totalBlastocyst cell

number

DOs 145 86.60.8a 7.71.6a 6.81.5a 89.14.1

DOs

163 92.6

0.8

b

26.2

1.6

b

22.6

1.3

b

92.8

3.9coDOs 142 93.81.9b 27.42.8b 25.93.0b 95.19.2coDOs 145 94.30.9b 27.33.1b 25.83.1b 102.17.2COCs 144 97.60.7c 36.43.3c 35.93.4c 99.45.7COCs 171 98.60.9c 39.11.9c 38.12.1c 106.56.3

Different superscripts (ac) within columns indicate statistical differences (P

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beginning of maturation, to 18 hr of incubation withFSH, while in absence of this hormone, only 10% ofhyaluronic acid is produced and cumulus expansiondoes not occur (Salustri et al., 1993). During themucification process, the secretion and assembly ofmatrix molecules by CCs are temporally regulated by

factors derived from both somatic cells and oocyte (Zhuoand Kimata, 2001). Further studies have identified anoocyte-secreted factor, growth differentiation factor-9(GDF-9), a member of TGF-b superfamily, which is ableto replace theoocyte to inducecumulus expansion(Elvinet al., 2000). It is noteworthy that GDF-9 knockout miceare completely unfertile, and folliculogenesis is arrestedto stages preceding cumulus expansion (Dong et al.,1996), strongly suggesting that GDF-9 is the oocytefactorthat controls thehyaluronic acid synthesisin CCs.

In conclusion, interactions between the cumulus-oophorus andthe oocyte are implicated in theregulationof female gamete development, meiotic maturation, and

oocyte-sperm interaction. Data from previous andpresent study support the central role played by CCsin the acquisition of the developmental competence ofthe oocyte also by promoting the accumulation of anadequate amount of GSH during IVM. However, theresults obtained with our culture system indicate thatthe direct interaction between oocyte and CCs is notnecessary during the maturation and fertilization pro-cesses and that presumablediffusible factor(s) producedby CCs and/or by the crosstalk between oocyte and CCsin the intact complex must be deeply investigated in thenear future.

Finally, since the removal of CCs from oocyte orzygotes at various stages of development still represents

a major limitation in female gamete manipulationtechniques in ART, this system could provide a technicalapproach to IVM and IVF of female gamete aftermanipulation and CC removal as well as in in vitroembryo production procedures of oocyte cryopreservedat the germinal vesicle stage (Modina et al., 2004).

ACKNOWLEDGMENTS

Authors thank Prof. C. Secchi, Prof. V. Borromeo, andDr. A. Berrini of the Biochemistry and Physiology Unit,Dept. of Veterinary Pathology, University of Milan, forhelp in GSH determination assay and Ms. Aru M. forher excellent technical support. A special thank goes to

Prof. John J. Peluso, University of Connecticut,USA, forhis critical suggestions to the manuscript.

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