Enhancement of developmental capacity of meiotically inhibited

Human Reproduction Vol.17, No.10 pp. 2706–2714, 2002

Enhancement of developmental capacity of meioticallyinhibited bovine oocytes by retinoic acid

P.Duque1, C.Dıez1, L.Royo1, P.L.Lorenzo2, G.Carneiro3, C.O.Hidalgo1, N.Facal1 andE.Gomez1,4

1SERIDA-CENSYRA, Camino de los Claveles, 604 Somio, 33203 Gijon, 2Departamento de Fisiologıa Animal, Facultad deVeterinaria, Universidad Complutense de Madrid, 28040, Spain and 3Department of Population Health and Reproduction, School ofVeterinary Medicine, University of California Davis, CA 94616, USA

4To whom correspondence should be addressed. E-mail: [email protected]

BACKGROUND: Although high vitamin A may be teratogenic to the embryo, retinol has been shown to supportoocyte developmental potential in vivo. Similarly, addition of retinol metabolite 9-cis-retinoic acid to in-vitro culturedoocytes could promote cytoplasmic maturation and subsequent early embryonic development. The objective of thisstudy was to evaluate the effects of 5 nmol/l retinoic acid during in-vitro pre-maturation and maturation of bovineoocyte–cumulus complexes. METHODS AND RESULTS: Oocytes were aspirated from cows and either kept undermeiotic arrest with 25 µmol/l roscovitine and matured, or allowed to mature in permissive conditions (control).Cortical granule migration was analysed both after pre-maturation and maturation by fluorescent labelling ofoocytes and subsequent laser confocal and fluorescence microscopy. Variables studied after in-vitro fertilization andculture in modified synthetic oviduct fluid were: (i) in-vitro embryonic development; (ii) ability of blastocysts tosurvive vitrification and warming; and (iii) differential cell counts measured by fluorescence microscopy. Althoughthe presence of 5 nmol/l retinoic acid throughout in-vitro maturation was harmful, its presence during pre-maturation alone improved cytoplasmic granular migration, embryonic development, cryopreservation tolerance,total cell numbers and, as a consequence, embryonic quality. CONCLUSIONS: Pre-maturation in the presence ofretinoic acid improves cytoplasmic competence of in-vitro matured bovine oocytes. Until more is known of themolecular mechanisms it would be irresponsible to use retinoic acid in maturation of human oocytes, especially inview of the narrow time window and possible species-specific differences in susceptibility and protection of theoocyte from epigenetic influences of retinol.

Key words: bovine/in-vitro/oocyte/retinoic/roscovitine

Introduction

Mammalian oocytes arrested at the diplotene stage of firstmeiotic prophase spontaneously resume meiotic maturationonce freed from their follicular environment. This process,involving germinal vesicle breakdown (GVBD), is independentfrom gonadotrophin stimulation (Edwards, 1965) and is typic-ally characterized by gradual chromatin condensation anddisapearance of a compact nucleolus and nuclear membrane(Kubelka et al., 1988). After meiotic progression and extrusionof the first polar body, the oocyte arrests at metaphase II andresumes activity at fertilization. In mammalian oocytes, themigration of the cortical granules (CG) is an important stepin cytoplasmic maturation and has been used as a criterion inthe assessment of maturity and organelle organization (Damianiet al., 1996). During maturation the majority of these CGmove from the site of production (usually the Golgi apparatus)to lie within a few micrometres of the plasma membrane (Cranand Esper, 1990). Successful fertilization depends on these

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changes, and most oocytes completing nuclear and cytoplasmicmaturation form a zygote after fertilization.

In-vitro procedures deprive the cultured oocyte of crucialin-vivo events such as the period of preovulatory development,during which the above-reported events occur rendering theoocyte developmentally competent (Bevers et al., 1997;Blondin et al., 1997; Hyttel et al., 1997). In this respect,in-vitro matured oocytes were found to show lower competencethan in-vivo matured oocytes in bovine (Van de Leemput et al.,1999) and human (Trounson et al., 2001). Although molecularevents regulating this are unknown, it is thought that theooplasm stores mRNA and proteins to provide maternal controlduring the first cleavages of embryonic development, beforethe embryonic genome is activated. Maintenance of reversiblemeiotic arrest in vitro at the germinal vesicle stage has beenfeasible in a number of species and, particularly in the bovine,by using a variety of chemicals interfering with the cAMPtransduction pathway (Sirard et al., 1998). The so-called pre-maturation period may permit cytoplasmic maturation to occur,

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Retinoic acid and meiotic inhibition

making the oocyte more developmentally competent (FouladiNashta et al., 1998). Moreover, this inhibition period provides achance to exogenously stimulate the oocyte cumulus–complex(COC). Recently it was shown that cattle oocytes could becultured under meiotic inhibition without decreasing theirresulting developmental potential (Mermillod et al., 2000).These authors used roscovitine, a potent inhibitor of M-phasepromoting factor (MPF) kinase activity, for a 24 h cultureperiod. Reversible roscovitine meiotic inhibition was alsodemonstrated in oocytes from other species such as pigs(Krischek and Meinecke, 2000; Marchal et al., 2001), Xenopuslaevis by microinjection (Flament et al., 2000), and Rhesusmonkey (Mitalipov et al., 2001). In contrast to untreatedoocytes, pregnancies at day 120 were obtained in cattle fromnuclear transfer oocytes incubated with roscovitine beforematuration (Kasinathan et al., 2001).

Vitamin A fulfils an essential role in the physiology ofvertebrates, being involved in cell growth and differentiation,embryonic development, and vision. The retinoids are a largefamily of natural and synthetic compounds related to vitaminA (all-trans-retinol). The vitamin A derivative retinoic acid(RA) is the most relevant retinoid during vertebrate develop-ment, but retinol is essential for pregnancy maintenance inmammals. All-trans-RA is reversibly converted to 9-cis-RAand other isomeres. These metabolites have the greatestbiological activity, although retinol acts directly for vision andspermatogenesis (Wellik et al., 1997), and possibly in placentaltissue (Sapin et al., 2000; Johansson et al., 2001). Both all-trans-RA and 9-cis-RA enter the cell nucleus and are able toactivate retinoic acid receptors (RAR), whereas retinoid Xreceptors (RXR) are activated only by 9-cis-RA (Mangelsdorfet al., 1994; Chambon, 1996). The RAR–RXR heterodimersare the functional units in transducing the retinoid signal atthe gene level. Recently, subtypes RARα, RARγ, RXRα andRXRβ, and retinaldehyde dehydrogenase 2 have been detectedin bovine embryos developed in vitro, from the oocyte tothe hatched blastocyst stage (Mohan et al., 2001, 2002).Immunostaining revealed the presence of proteins for RARαand RARγ-2 (Mohan et al., 2001) and RXRβ (Mohan et al.,2002) in the trophectoderm (TE) and the inner cell mass (ICM)in intact and hatched blastocysts. Both vitamin A deficiencyand high concentrations of retinoid are associated with develop-mental abnormalities, through altering the normal relationshipbetween cellular retinoid levels and the embryonic geneticdevelopmental programme (Morris-Kay and Ward, 1999).Whereas it has been suggested that the requirement for vitaminA activity in the embryo begins at the time of first organinitiation, but not earlier (Zile, 2001), there is evidence thatthe oocyte’s developmental competence could be enhanced byretinoid support during oocyte intrafollicular growth. In fact,retinol administration to donor animals improved embryonicquality in both superovulated cows (Shaw et al., 1995) andsheep (Eberhardt et al., 1999), and in non-superovulated gilts(Whaley et al., 1997, 2000). In addition, more oocytes andembryos were obtained in response to superovulating rabbitsthat had higher blood levels of vitamin A (Besenfelder et al.,1996). Recently, it has been demonstrated that the yield ofoocytes increased in donor cows injected with retinol (Hidalgo

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et al., 2002), and blastocyst development and quality arehigher for COC matured in the presence of RA (Duque et al.,2002). Cytoplasmic retinol-binding proteins are found in cow(Mohan et al., 2001) and rat (Wardlaw et al., 1997) oocytes,and nearby granulosa cells in rats (Wardlaw et al., 1997) andin human ovaries (Ong and Page, 1986) where they specificallybind retinol and regulate its effects

The beneficial effect of vitamin A during oocyte growth invivo can be reproduced by retinol derivatives, added to anin-vitro culture system into which the oocytes are meioticallyarrested. Since RA acts on cells to establish or change thepattern of gene activity, this retinoid could influence cyto-plasmic maturation and the subsequent capacity of the oocyteto progress in development. Apart from the oocyte itself, theinfluence of the RA could be exerted through cumulus–granulosa cells. To date, no studies on the combined effect ofRA on oocyte cytoplasmic and nuclear maturation have beenreported. Thus, in the present work the effect of 9-cis-RAduring pre-maturation and maturation within the OCC wasanalysed. Parameters evaluated were oocyte cytoplasmic granu-lar migration, blastocyst development and blastocyst quality,measured as trophectoderm and inner mass cell numbers andthe ability to survive cryopreservation.

Materials and methodsAll chemicals were purchased from Sigma (Madrid, Spain), unlessotherwise indicated.

Collection of COC

Ovaries recovered from slaughtered Asturiana de los Valles cowswere placed in NaCl solution (9 mg/ml) containing antibiotics(penicillin, 100 IU/ml and streptomycin sulphate, 100 µg/ml) andmaintained at 25–30°C until COC collection. Ovaries were washedtwice in distilled water and once in freshly prepared saline. Visiblefollicles 2–7 mm in size were aspirated through an 18-gauge needleconnected to a syringe, and the contents recovered into a 50 mlCorning tube. Follicular fluid and COC were placed in an ovumconcentrating device (Comextrade, Tarragona, Spain) and rinsed threetimes with holding medium (HM: TCM199; Invitrogen, Barcelona,Spain; 25 mmol/l HEPES and bovine serum albumin 0.5 mg/ml)supplemented with 2 IU/ml of heparin.

In-vitro pre-maturation, maturation and experimental design

Oocytes enclosed in a compact cumulus with evenly granulatedcytoplasm were selected and washed for three times in HM. ThoseCOC assigned to groups undergoing pre-maturation were washedtwice in basic medium (BM: TCM199 � PVA 0.5 mg/ml) containingroscovitine 25 µmol/l, which was previously dissolved in DMSOand stored in aliquots at –20°C until use, as previously described(Mermillod et al., 2000). Pre-maturation was performed by culturingCOC in BM with roscovitine 25 µmol/l during 24 h. Those COCcultured under permissive maturation conditions were washedthree times in HM and twice in BM. Maturation was carried outin BM containing pFSH (1 µg/ml), LH (5 µg/ml) and 17β-estradiol(1 µg/ml) for 24 h. Prematured COC were allowed to maturefor 24 h under permissive, non-meiotically inhibiting conditions.Incubation were performed in 4-well dishes (Nunc, Biocen, Spain)containing 500 µl of culture medium at 39°C in 5% CO2 under airand high humidity. COC were treated in the presence and absence of9-cis-RA 5 nmol/l during pre-maturation and/or maturation, according


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Table I. Cortical granule (CG) distribution and nuclear maturation in bovine oocytes during pre-maturationand maturation cultures

Group Labelled Pre-maturation Maturation CG migration Dominant nuclearoocytes 9-cis RA (�/–) pattern status

1 10 No No 1 GV2 6 No (�) 2 MII3 6 (�) No 1 GV4 6 (�) No 2 GV5 5 (�) (�) 3 MII6 5 (�) (�) 3 MII7 7 (�) (�) 3 MII8 7 (�) (�) 4 MII

GV � germinal vesicle; MII � metaphase II.

to a 2�2 factorial design. A group of non-inhibited COC acted asa control.

Labelling of oocytes with fluoroscein isothiocianate (FITC) lectinsand CG and chromosomal staining

A number of oocytes (immature, prematured and/or matured oocyteswith or without RA) representative of each treatment were processed.After removal of surrounding cumulus cells with a narrow glasspipette, the zona pellucida was removed with 0.1% pronase. Zona-free oocytes were washed three times, fixed with 2% paraformaldehydein phosphate-buffered saline (PBS) for at least 12 h in a 35 mm dishat 5°C, and washed four times in blocking solution (PBS containingsodium azide and 100 mmol/l glycine). Oocytes in blockingsolution were incubated in 10 µg/ml FITC-labelled Lens culinarisagglutinin (LCA, FL-1041; Vector Labs, Inc., Burlingame, CA, USA)for 15 min in the dark. Chromatin was stained with 10 µg/mlpropidium iodide for 5 min. After staining, oocytes were washed andmounted between a coverslip and a glass slide supported by silicone(Lorenzo et al., 1994) with antifade mounting medium (Vectashield;Vector Laboratories, Inc.), and the coverslip was sealed with nailpolish. Samples were examined using laser-scanning confocalmicroscopy.

Fluorescence microscopy

Laser-scanning confocal microscopy was performed using a Bio-RadMRC 1024 ES equipped with a Krypto-argon ion laser for thesimultaneous excitation of fluorescein for CG and propidium iodidefor DNA (488, laser line and 680 DF 32 respectively). Oocytesserving as controls were treated using the same procedure and wereincubated in blocking solution without FITC-labelled LCA. Imageswere recorded digitally and archived on an erasable magnetic opticaldisk. Typical examples of the CG distribution in the oocytes areshown in Figure 1.

In-vitro fertilization

Sperm separation was carried out using a swim-up procedure similar tothat reported elsewhere (Parrish et al., 1986). Briefly, semen fromone frozen straw of a single bull of proven fertility was thawedin a water bath and added to a polystyrene tube containing 1 ml ofpre-equilibrated sperm–Tyrode–albumin–lactate–pyruvate (Sperm-TALP). After 1 h of incubation, ~700 µl of the upper layer ofsupernatant containing the motile sperm was removed. The spermwere centrifuged for 7 min at 100 g and the supernatant aspirated toleave a pellet of ~100 µl in volume. Sperm concentration wasdetermined with a haemocytometer. After 22–24 h of maturation, theCOC were washed twice in holding medium and placed in four-well culture dishes containing pre-equilibrated fertilization

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medium (Fert-TALP) with heparin (10 µg/ml, Calbiochem, LaJolla, CA, USA). Spermatozoa were then added at a concentrationof 2�106 cells/ml in 500 µl of medium per well containing �100COC. IVF was accomplished by incubating oocytes and sperm cellstogether for 18–20 h at 39°C in 5% CO2 under high humidity.

Embryo culture

Presumptive zygotes were vortexed for 2 min to separate cumuluscells and washed three times in HM and once in culture medium.Embryo culture was performed in modified synthetic oviduct fluid(SOF) containing amino acids, citrate and myoinositol (Holm et al.,1999) adjusted to 285 mOsm and pH 7.2–7.3. Fetal calf serum (FCS)was added at 42 h post-insemination (PI). Droplets of culture medium(1–2 µl/embryo) were prepared in four-well dishes under mineral oiland equilibrated for 2 h before addition of zygotes. Incubations werecarried out at 39°C in 5% CO2, 5% O2 and 90% N2. Culture mediawere renewed at 66 h (day 3) and 138 h (day 6) PI, and embryonicdevelopment was recorded on days 3, 6, 7 and 8.

Vitrification and warming

Expanded blastocysts (day 7) were kept in 10% glycerol in ovumculture medium (OCM) with 20% FCS for 5 min. Blastocysts werethen incubated in 20% glycerol � 10% ethylene glycol in OCM with20% FCS. Finally, samples were washed in 25% glycerol � 25%ethylene glycol in OCM with 20% FCS (Kaidi et al., 2000).Embryos were loaded into 0.25 ml straws between two columns ofa 0.85 mol/l galactose solution in OCM separated from the embryosby air bubbles. The straws were placed in liquid nitrogen vapour for2 min before being plunged into liquid nitrogen. The vitrified embryoswere warmed for 5 s in air and 10 s in a water bath at 30°C. Thecontents of the straws were expelled into a Petri dish and embryoswere mixed in the galactose solution by slight agitation. After 5 min,embryos were transferred to OCM with 20% FCS during 5 min.Warmed embryos were washed in Menezo-B2 medium � 5% FCSand co-cultured in 4-well dishes (500 µl) on a confluent monolayerof Vero cells at 39°C, 5% CO2 in air and high humidity.

Blastocysts differential cell counting

Cells were counted in day 8 hatched and expanded blastocysts aspreviously described (Van Soom et al., 1996). Briefly, expandedembryos were incubated in PBS � 5% pronase for 1 min and in acidTyrode’s solution for 1 min in order to remove the zona pellucida.

Once devoid of the zona pellucida, expanded and hatched blasto-cysts were incubated in trinitrobenzenesulphonic acid and rabbitantiserum (antiDNP-BSA) solution. Thereafter, the blastocysts wereincubated in guinea-pig complement serum for 30 min at 39°C.Embryos were subsequently washed in TCM199 HEPES � 10 µl/ml



Figure 1. Confocal microscopic images of bovine oocytes after staining with fluorescein isothiocyanate–Lens culinaris agglutinin andpropidium iodide showing the pattern of cortical granules distribution (a–d; green) and chromatin configuration (e and d; red). Imagespresent equatorial sections of each oocyte.

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P.Duque et al.

propidium iodide. Samples were fixed in ethanol and incubated inbisbenzimide (Hoechst 33342 in 10 µl/ml ethanol). Finally, aftermounting on a glass slide the samples were evaluated under afluorescence microscope at �400 with an excitation filter of 330–385 nm and barrier filter of 420 nm. TE fluoresce red and ICMappear blue.

Statistical analysis

The data on development and survival rate (percentage) after cryo-preservation were arcsin transformed. Data were submitted to one-way ANOVA and Duncan’s test for means, and expressed as mean� SEM (development, cryopreservation, and cell numbers).

Results

Cytoplasmic maturation study

Forty-five out of 52 oocytes used in the cytoplasmicmaturation study were examined for CG localization andmigration patterns. The remaining oocytes were lost duringthe staining procedures. The stage, treatment and numbers oflabelled oocytes were coded as shown in Table I. Figure 1a–fshows CG localization and nuclear stage structures after pre-maturation and/or maturation periods. Four patterns of CGdistribution were found: pattern 1: oocytes with CG distributedas clusters only in the central medullary zone (Figure 1a);pattern 2: oocytes with CG located between the oolemma andthe medullary zone (Figure 1b); pattern 3: oocytes in which CGmigrated completely and formed clusters lining the oolemma(Figure 1c); and pattern 4: oocytes in which CG formed amonolayer lining the oolemma (Figure 1d).

Pattern 1 was typically found in immature oocytes (group1) and in oocytes at the end of the pre-maturation period inthe absence of RA (group 3). However, oocytes examinedafter pre-maturation with RA (group 4) presented a markedalthough incomplete migration in contrast to groups 1 and 3,which showed CG in the medullary zona. All groups of oocytesabove showed nuclear germinal vesicle configuration (notshown as a Figure). Oocytes matured in permissive conditions(group 2) showed partial migration (pattern 2), with CGclusters located between the oolemma and the medullary zone.When oocytes which were prematured in the absence of RAwere allowed to mature, both in the presence (group 5) andin the absence (group 6) of RA, complete CG migration tothe cortex was noted (pattern 3). This pattern is also presentin oocytes prematured and matured with RA (group 7).

Interestingly, the presence of RA led to oocytes exhibitingCG migration at the end of pre-maturation (group 4). Inaddition, oocytes from group 8 showed less clustering thanthe other groups analysed, and their CG formed a uniformmonolayer beneath the oolemma (pattern 4). This monolayer,seen in six out of seven oocytes analysed, was absent in othergroups, which suggests that the presence of RA during pre-maturation improves granular migration. Pictures frommetaphase I and II can be seen in Figure 1e and f respectively.

Development, cryopreservation survival and cell counts

As seen in Table II, the rates of morulae and blastocystsfrom oocytes treated with RA during roscovitine-induced

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pre-maturation but not during subsequent maturation, werecomparable with those from uninhibited oocytes whereas theremaining groups developed at lower rates. Proportions ofembryos at the 5–8-cell stage (not shown in Tables) werehigher for uninhibited oocytes (64.9 � 4.1) than for theirprematured counterparts (values comprised between 42.9 �5.1 and 45.3 � 7.7; P � 0.05). Treatment with RA duringpre-maturation or maturation alone increased blastocystdevelopment rates at 4 h post-warming (Table III). However,blastocysts derived from a pre-maturation treatment with RAsurvived longer as compared with other groups and showedimproved hatching. Survival differences observed at 72 h werethe same as at 48 h. In Table IV, oocytes treated with RA atany time tended to give blastocysts having higher ICMcounts, and RA during pre-maturation alone increased totalcell numbers. Embryos derived from oocytes exposed toRA during pre-maturation and maturation showed a strikingreduction in TE cell numbers, which demonstrates an abnormalcell distribution.

Discussion

The nuclear maturation programme of the oocyte resumes afteroocyte removal from the follicle. Transcription is inhibited oncemeiosis resumes, which restricts the molecular programmingrequired by the oocyte to develop. The purpose of inhibitingmeiosis in vitro is to allow for stable mRNA and proteinaccumulation in the cytoplasm (Kastrop et al., 1990; Pavloket al., 1997, 2000). Once stored, these factors will allow theoocyte to acquire development potential, which is the majordifference between in-vitro and in-vivo matured oocytes. Therole of retinoids during preblastulation development is widelyunexplored both in vivo and in vitro, and during in-vitromaturation. Retinoids, especially RA, can induce differentiationin a number of cell systems and play a role in controllingevents during the cell cycle, as the MAP kinase phosphatasegene, X17C, is RA inducible (Old et al., 1995). According tothe current literature, RA can induce or silence the expressionof hundreds of genes, though little is known of the normalchain of events within an intact tissue.

Pre-maturation in the presence of RA improves cytoplasmiccompetence of in-vitro matured bovine oocytes from COC.Upon collection, CG are distributed in the cytoplasm of oocytesat the GV stage. As maturation proceeds, the granules migrateto the cortex and occupy the area just beneath the oolemma,at same time the nucleus enters MII stage. Cortical granulemigration is a common phenomenon in mammalian oocytesduring maturation both in vivo and in vitro (Yanagimachi,1994; Wang et al., 1997a). This migration is associated witha gain in developmental competence by the oocyte (Hosoeand Shioya, 1997) and blocks polyspermy once migrated CGcontents are released (Hyttel et al., 1988; Wang et al., 1997b;Nagano et al., 1999). The most relevant finding within ourCG migration study was probably that RA induced CGmigration prior to maturation. Also, the CG distribution afterRA exposure formed a uniform monolayer beneath theoolemma with lesser clustering once RA-prematured oocyteswere allowed to mature in the absence of RA. Taken together,



Table II. Effect of the presence (�) or the absence (�) of 9-cis-retinoic acid during roscovitine-mediated meiotic inhibition during in-vitro pre-maturation(PM) and maturation (M) of bovine oocyte–cumulus complexes on subsequent embryonic development

(9-cis-retinoic) n Cleaved Morulae Blastocysts

PM M Day 6 Day 7 Day 8 Expanded

(�) (�) 215 79.7 � 2.3 31.1 � 5.1c 4.9 � 1.3c 17.4 � 3.4b 19.3 � 3.1b 11.5 � 3.7b

(�) (�) 211 79.0 � 5.1 28.5 � 3.6c 6.9 � 1.2bc 15.0 � 4.7b 20.3 � 2.9b 11.8 � 1.7b

(�) (�) 225 81.0 � 2.9 40.5 � 3.9b 11.8 � 3.3ab 23.6 � 4.6 28.2 � 3.3a 21.3 � 2.3a

(�) (�) 199 83.4 � 2.8 29.3 � 4.4c 5.7 � 2.3c 17.4 � 2.7b 18.0 � 2.5b 13.5 � 3.3b

No (�) 205 87.4 � 4.1 54.9 � 3.7a 15.2 � 1.3a 29.3 � 3.4a 32.7 � 3.2a 25.0 � 1.8a

Data are percentages of matured oocytes � SEM (six replicates).n � numbers of cultured oocytes.a,b,cDifferent superscripts show significant differences (P � 0.05).

Table III. Post-warming development of vitrified day 7 bovine expanded blastocysts derived from oocyte–cumulus complexes treated (�) or untreated (�)with 9-cis-retinoic acid during roscovitine-mediated meiotic inhibition (pre-maturation, PM) and/or during maturation (M)

(9-cis-retinoic) 4 h 24 h 48 h

PM M n Living Hatched Living Hatched Living Hatched

(�) (�) 11 77.5 � 13a 0.0b 17.5 � 11 0.0b 12.5 � 12 0.0(�) (�) 9 50.0 22 0.0b 30.0 � 20 0.0b 0.0b 0.0(�) (�) 21 75.0 � 7a 12.5 � 12a 44.8 � 13 15.6 � 12a 21.9 � 7a 7.3 � 4(�) (�) 16 44.4 � 16 4.2 � 4 5.5 � 5.5 0.0b 5.5 � 5 5.5 � 5No (�) 34 39.0 � 13b 1.4 � 1b 26.5 � 6 1.4 � 1b 11.5 � 6 2.7 � 1

Data are percentages of matured oocytes � SEM from a minimum of three replicates per treatment.n � number of cultured thawed embryos.a,bDifferent superscripts show significant differences (P � 0.05).

Table IV. Effect of the presence (�) or the absence (�) of 9-cis-retinoic acid during roscovitine-mediatedmeiotic inhibition (pre-maturation, PM) and/or during maturation (M) of bovine oocyte–cumulus complexesupon differential cell counts of day 8 expanded blastocysts

(9-cis-retinoic) No. of cells � SEM PercentageICM/Total

PM M n ICM TFE Total

(�) (�) 7 27.2 � 5.6 65.9 � 7.3a 93.1 � 11.3 28.5 � 3.9b

(�) (�) 10 26.4 � 3.2 39.1 � 7.5b 65.6 � 8.9b 42.8 � 6.1a

(�) (�) 8 24.9 � 6.9 73.1 � 4.5a 100.2 � 5.2a 24.1 � 5.8b

(�) (�) 7 20.4 � 3.5 71.7 � 11.4a 92.1 � 14.0 23.0 � 2.2b

No (�) 8 17.1 � 1.5 55.2 � 10.1 72.3 � 9.8b 26.5 � 3.8b

Embryos counted (n) were from five replicates.a,b,cDifferent superscripts show significant differences (P � 0.05).ICM � inner cell mass; TFE � trophectoderm.

these results suggest a role for RA in the improvement ofdevelopmental competence. However, the exact timing (andpossibly also the concentration) of RA exposure is criticalsince it alters the normal CG migration and distribution. Thearea devoid of granules immediately overlying the secondmeiotic spindle (Szollosi, 1967; Nicosia et al., 1977) has beencalled the CG-free domain (CGFD) (Ducibella et al., 1988).Development of the CGFD in hamster and mouse oocytes isa result of local exocytosis and redistribution of CG (Okadaet al., 1986; Ducibella et al., 1988). However, as previouslydescribed in the bovine (Long et al., 1994), no obvious CGFDwas observed in oocytes in the current experiment duringmeiotic maturation. Similar to observations in the mouse

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(Szollosi, 1967) we did not observe migration of CG tothe deeper cytoplasmic portion in the bovine. As reportedpreviously, bovine CG organization becomes discontinuous incomparison with horse oocytes (Wang et al., 1997b).

Embryo quality may be assessed by the number of embryoniccells (Ellington et al., 1990), and in-vitro produced blastocystsare often selected for freezing or vitrification according toquality. In-vitro re-expansion and hatching using somaticcell co-culture after cryopreservation is a common test forembryonic viability. In the present work, day 7 embryosbelonging to groups having the highest total cell numberson day 8 survived better to post-warming using co-culture.However, ICM cells and the ratio ICM/trophectoderm cells


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varied without affecting in-vitro survival and hatching, whichhas been described elsewhere (Thompson et al., 1995; VanSoom et al., 1996). The best criterion for viability is the birthof normal calves, which does not always equate with highembryo survival rates after thawing (Van Wagtendonk-deLeeuw et al., 1995; Donnay et al., 1998). In the present work,RA exerted a proliferative effect which was not evidentduring maturation of bovine oocytes exposed to meiotic arrest,regardless of whether RA was present during pre-maturationor not. Furthermore, prolonged exposure of COC to RA causedembryos to have an abnormally low total cell number, adisproportionate embryonic cell distribution and reduced abilityto survive cryopreservation, which could be interpreted as alow embryonic viability. The reduction in cell numbers onlyaffected trophoblast cells, the ICM having a cell number rangecomparable to normal. Interestingly, the combined effects ofpre-maturation together with the presence of RA during thepre-maturation period only increased trophectoderm cell countsand, as a consequence, total cell numbers in blastocysts. Inaddition, the number of cells allocated to the ICM had atendency to increase in those blastocysts derived from oocytestreated with RA, in accordance with previous observations(Duque et al., 2002) on blastocyst development and survivalafter vitrification and warming. Our results are not inconsistentwith previous data showing that the presence of RA duringnon-inhibited bovine IVM improves blastocyst development(S.Ikeda, personal communication; Duque et al., 2002) andincreases numbers and the proportions of cells allocated to theICM (Duque et al., 2002). The influence of RA on GV-arrestedoocytes could be correlated to weak [3H]uridine labelling ofthe GV area (Pavlok et al., 2000) together with incompleteinactivation of hnRNA synthesis (Hyttel et al., 1997) at thisstage. RA may thus contribute to direct or indirect epigeneticprocesses influencing developmental capacity.

The detrimental effect of a RA overexposure (i.e. thepresence of RA during both pre-maturation and maturation),or the neutral effect observed during maturation alone, mayrely on an interaction of RA with FSH. As a consequence,RA diminishes FSH-induced expression of LH receptor inporcine (Hattori et al., 2000) and rat (Minegishi et al., 2000)granulosa cells. Nevertheless, when porcine granulosa cellsare treated with RA and washed before addition of FSH andLH, the expression of LH receptor mRNA is not reversed(Hattori et al., 2000). Following the proposed model, treatingCOC with RA during pre-maturation alone would permit theexpression of LH receptor within the cumulus cells. Althoughin our work concomitant FSH and RA was not beneficial,neither was a deleterious effect was observed.

Our preliminary observations suggests an involvement ofmidkine in RA effects. This influence is currently quantitativelyinvestigated. Midkine has been isolated in bovine follicularfluid (Ohyama et al., 1994), and in rat granulosa cells isdependent on gonadotrophins (Karino et al., 1995; Minegishiet al., 1996). Since simultaneous addition of FSH and RA toa granulosa cell culture did not significantly change theaccumulation of midkine mRNA observed with either FSH orRA alone (Minegishi et al., 1996), this effect could beassociated with the suppression of FSH receptor by RA

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(Minegishi et al., 1996, 2000). Recombinant midkine enhanceddevelopment to the blastocyst stage of bovine oocytes, itseffect being mainly mediated by cumulus–granulosa cellsduring in-vitro maturation (Ikeda et al., 2000a,b). The need toensure the supply of retinoids to the embryos within aphysiological range makes the choice of dosage for retinoidsa delicate matter. As improved blastocyst development andquality was obtained with 5 nmol/l RA under permissivematuration conditions (Duque et al., 2002), we adopted thisconcentration in our experiments. Some other reports of theeffects of RA on granulosa cells were also considered. Amongthem RA resulted in a 2-fold increase in midkine mRNAlevels (0.3 µmol/l) (Minegishi et al., 1996) and altered LHreceptor mRNA expression (1 µmol/l) (Hattori et al., 2000).The retinol concentration in human serum is 1–2 µmol/l, andfetal bovine serum contains �20 nmol/l (Lane et al., 1999).Improved blastocyst development was obtained with all-trans-RA 1 µmol/l (S.Ikeda, personal communication). This isomerehas been reported to be 25 times less potent than 9-cis-RA(Thaller et al., 1993). In the present study a marked responsewas obtained by overexposure of bovine oocytes to retinoids.These detrimental effects could be related to a well documentedretinoid imbalance associated with developmental abnormality(Morriss-Kay and Ward, 1999). Again, midkine could be anintermediary in these developmental phenomena (Griffith andZile, 2000). In fact, both excess and deficiency of retinoidscause abundant teratogenic defects due to its pleiotrophicactivity. An analogous detrimental effect has been suggestedwith oocytes matured with RA recovered from cows treatedwith retinol (Hidalgo et al., 2002).

Expression of the cellular retinoic acid-binding protein II(CRABP II) has been found in the rat, which is confined togranulosa cells from mature follicles and luteal cells (Buccoet al., 1995; Wardlaw et al., 1997; Zheng et al., 1999), butnot found in other species. CRABP control the access of RAto the cell nucleus protecting against the RA excess. The lateintrafollicular expression of CRABP II could mean that thisperiod is more sensitive to RA and needs protection. However,immature bovine oocyte and granulosa cells might show abetter tolerance to, and/or a higher dependence on, RA, asthey lack a system to regulate the retinoid levels. This reinforcesthe need of keeping RA within physiological levels in vitro,to perform RA dose–response studies and to investigate theexpression of retinoid binding proteins in the bovine follicle.

Until more is known about the molecular processes, targetsand regulation of RA in the follicle and oocyte in experimentalsystems such as those presented in this study, it appearspremature and irresponsible to use RA in maturation ofhuman oocytes, especially in view of the narrow time windowand possible species-specific differences in susceptibility andprotection ot the oocyte from epigenetic influences of retinol.This is important with respect to the potentially teratogenicand irreversible long-term effects of RA resulting in congenitalabnormalities during embryogenesis and unknown alterationsin gene expression even later in life.

AcknowledgementsWe wish to thank Dr Felix Goyache for criticisms, Dr Irwin K.M.Liufor support and Frank Ventimiglia for his valuable technical help.Grant support: CICYT-FEDER (project 1FED97-0023).



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Submitted on May 2, 2002; resubmitted on March 14, 2002; accepted on May24, 2002


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Enhancement of developmental capacity of meiotically inhibited