1 SERIDA-CENSYRA, Camino de los Claveles, 604 Somió, 33203 Gijón, 2 Departamento de Fisiología Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Spain and 3 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California Davis, CA 94616, USA
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Abstract |
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Key words: bovine/in-vitro/oocyte/retinoic/roscovitine
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Introduction |
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In-vitro procedures deprive the cultured oocyte of crucial in-vivo events such as the period of preovulatory development, during which the above-reported events occur rendering the oocyte 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 competence than in-vivo matured oocytes in bovine (Van de Leemput et al., 1999
) and human (Trounson et al., 2001
). Although molecular events regulating this are unknown, it is thought that the ooplasm stores mRNA and proteins to provide maternal control during the first cleavages of embryonic development, before the embryonic genome is activated. Maintenance of reversible meiotic arrest in vitro at the germinal vesicle stage has been feasible in a number of species and, particularly in the bovine, by using a variety of chemicals interfering with the cAMP transduction pathway (Sirard et al., 1998
). The so-called pre-maturation period may permit cytoplasmic maturation to occur, making the oocyte more developmentally competent (Fouladi Nashta et al., 1998
). Moreover, this inhibition period provides a chance to exogenously stimulate the oocyte cumuluscomplex (COC). Recently it was shown that cattle oocytes could be cultured under meiotic inhibition without decreasing their resulting developmental potential (Mermillod et al., 2000
). These authors used roscovitine, a potent inhibitor of M-phase promoting factor (MPF) kinase activity, for a 24 h culture period. Reversible roscovitine meiotic inhibition was also demonstrated in oocytes from other species such as pigs (Krischek and Meinecke, 2000
; Marchal et al., 2001
), Xenopus laevis by microinjection (Flament et al., 2000
), and Rhesus monkey (Mitalipov et al., 2001
). In contrast to untreated oocytes, pregnancies at day 120 were obtained in cattle from nuclear transfer oocytes incubated with roscovitine before maturation (Kasinathan et al., 2001
).
Vitamin A fulfils an essential role in the physiology of vertebrates, being involved in cell growth and differentiation, embryonic development, and vision. The retinoids are a large family of natural and synthetic compounds related to vitamin A (all-trans-retinol). The vitamin A derivative retinoic acid (RA) is the most relevant retinoid during vertebrate development, but retinol is essential for pregnancy maintenance in mammals. All-trans-RA is reversibly converted to 9-cis-RA and other isomeres. These metabolites have the greatest biological activity, although retinol acts directly for vision and spermatogenesis (Wellik et al., 1997), and possibly in placental tissue (Sapin et al., 2000
; Johansson et al., 2001
). Both all-trans-RA and 9-cis-RA enter the cell nucleus and are able to activate retinoic acid receptors (RAR), whereas retinoid X receptors (RXR) are activated only by 9-cis-RA (Mangelsdorf et al., 1994
; Chambon, 1996
). The RARRXR heterodimers are the functional units in transducing the retinoid signal at the gene level. Recently, subtypes RAR
, RAR
, RXR
and RXRß, and retinaldehyde dehydrogenase 2 have been detected in bovine embryos developed in vitro, from the oocyte to the hatched blastocyst stage (Mohan et al., 2001
, 2002
). Immunostaining revealed the presence of proteins for RAR
and RAR
-2 (Mohan et al., 2001
) and RXRß (Mohan et al., 2002
) in the trophectoderm (TE) and the inner cell mass (ICM) in intact and hatched blastocysts. Both vitamin A deficiency and high concentrations of retinoid are associated with developmental abnormalities, through altering the normal relationship between cellular retinoid levels and the embryonic genetic developmental programme (Morris-Kay and Ward, 1999
). Whereas it has been suggested that the requirement for vitamin A activity in the embryo begins at the time of first organ initiation, but not earlier (Zile, 2001
), there is evidence that the oocytes developmental competence could be enhanced by retinoid support during oocyte intrafollicular growth. In fact, retinol administration to donor animals improved embryonic quality in both superovulated cows (Shaw et al., 1995
) and sheep (Eberhardt et al., 1999
), and in non-superovulated gilts (Whaley et al., 1997
, 2000
). In addition, more oocytes and embryos were obtained in response to superovulating rabbits that had higher blood levels of vitamin A (Besenfelder et al., 1996
). Recently, it has been demonstrated that the yield of oocytes increased in donor cows injected with retinol (Hidalgo et al., 2002
), and blastocyst development and quality are higher 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
) and in human ovaries (Ong and Page, 1986
) where they specifically bind retinol and regulate its effects
The beneficial effect of vitamin A during oocyte growth in vivo can be reproduced by retinol derivatives, added to an in-vitro culture system into which the oocytes are meiotically arrested. Since RA acts on cells to establish or change the pattern of gene activity, this retinoid could influence cytoplasmic maturation and the subsequent capacity of the oocyte to progress in development. Apart from the oocyte itself, the influence of the RA could be exerted through cumulusgranulosa cells. To date, no studies on the combined effect of RA on oocyte cytoplasmic and nuclear maturation have been reported. Thus, in the present work the effect of 9-cis-RA during pre-maturation and maturation within the OCC was analysed. Parameters evaluated were oocyte cytoplasmic granular migration, blastocyst development and blastocyst quality, measured as trophectoderm and inner mass cell numbers and the ability to survive cryopreservation.
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Materials and methods |
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Collection of COC
Ovaries recovered from slaughtered Asturiana de los Valles cows were placed in NaCl solution (9 mg/ml) containing antibiotics (penicillin, 100 IU/ml and streptomycin sulphate, 100 µg/ml) and maintained at 2530°C until COC collection. Ovaries were washed twice in distilled water and once in freshly prepared saline. Visible follicles 27 mm in size were aspirated through an 18-gauge needle connected to a syringe, and the contents recovered into a 50 ml Corning tube. Follicular fluid and COC were placed in an ovum concentrating device (Comextrade, Tarragona, Spain) and rinsed three times 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 granulated cytoplasm were selected and washed for three times in HM. Those COC assigned to groups undergoing pre-maturation were washed twice in basic medium (BM: TCM199 + PVA 0.5 mg/ml) containing roscovitine 25 µmol/l, which was previously dissolved in DMSO and stored in aliquots at 20°C until use, as previously described (Mermillod et al., 2000). Pre-maturation was performed by culturing COC in BM with roscovitine 25 µmol/l during 24 h. Those COC cultured under permissive maturation conditions were washed three times in HM and twice in BM. Maturation was carried out in BM containing pFSH (1 µg/ml), LH (5 µg/ml) and 17ß-estradiol (1 µg/ml) for 24 h. Prematured COC were allowed to mature for 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 air and high humidity. COC were treated in the presence and absence of 9-cis-RA 5 nmol/l during pre-maturation and/or maturation, according to a 2x2 factorial design. A group of non-inhibited COC acted as a control.
Labelling of oocytes with fluoroscein isothiocianate (FITC) lectins and CG and chromosomal staining
A number of oocytes (immature, prematured and/or matured oocytes with or without RA) representative of each treatment were processed. After removal of surrounding cumulus cells with a narrow glass pipette, the zona pellucida was removed with 0.1% pronase. Zona-free oocytes were washed three times, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for at least 12 h in a 35 mm dish at 5°C, and washed four times in blocking solution (PBS containing sodium azide and 100 mmol/l glycine). Oocytes in blocking solution were incubated in 10 µg/ml FITC-labelled Lens culinaris agglutinin (LCA, FL-1041; Vector Labs, Inc., Burlingame, CA, USA) for 15 min in the dark. Chromatin was stained with 10 µg/ml propidium iodide for 5 min. After staining, oocytes were washed and mounted 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 nail polish. Samples were examined using laser-scanning confocal microscopy.
Fluorescence microscopy
Laser-scanning confocal microscopy was performed using a Bio-Rad MRC 1024 ES equipped with a Krypto-argon ion laser for the simultaneous excitation of fluorescein for CG and propidium iodide for DNA (488, laser line and 680 DF 32 respectively). Oocytes serving as controls were treated using the same procedure and were incubated in blocking solution without FITC-labelled LCA. Images were recorded digitally and archived on an erasable magnetic optical disk. Typical examples of the CG distribution in the oocytes are shown in Figure 1.
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Embryo culture
Presumptive zygotes were vortexed for 2 min to separate cumulus cells 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 7.27.3. Fetal calf serum (FCS) was added at 42 h post-insemination (PI). Droplets of culture medium (12 µl/embryo) were prepared in four-well dishes under mineral oil and equilibrated for 2 h before addition of zygotes. Incubations were carried out at 39°C in 5% CO2, 5% O2 and 90% N2. Culture media were renewed at 66 h (day 3) and 138 h (day 6) PI, and embryonic development was recorded on days 3, 6, 7 and 8.
Vitrification and warming
Expanded blastocysts (day 7) were kept in 10% glycerol in ovum culture medium (OCM) with 20% FCS for 5 min. Blastocysts were then incubated in 20% glycerol + 10% ethylene glycol in OCM with 20% 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 of a 0.85 mol/l galactose solution in OCM separated from the embryos by air bubbles. The straws were placed in liquid nitrogen vapour for 2 min before being plunged into liquid nitrogen. The vitrified embryos were warmed for 5 s in air and 10 s in a water bath at 30°C. The contents of the straws were expelled into a Petri dish and embryos were 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 Ménézo-B2 medium + 5% FCS and co-cultured in 4-well dishes (500 µl) on a confluent monolayer of 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 as previously described (Van Soom et al., 1996). Briefly, expanded embryos were incubated in PBS + 5% pronase for 1 min and in acid Tyrodes solution for 1 min in order to remove the zona pellucida.
Once devoid of the zona pellucida, expanded and hatched blastocysts were incubated in trinitrobenzenesulphonic acid and rabbit antiserum (antiDNP-BSA) solution. Thereafter, the blastocysts were incubated in guinea-pig complement serum for 30 min at 39°C. Embryos were subsequently washed in TCM199 HEPES + 10 µl/ml propidium iodide. Samples were fixed in ethanol and incubated in bisbenzimide (Hoechst 33342 in 10 µl/ml ethanol). Finally, after mounting on a glass slide the samples were evaluated under a fluorescence microscope at x400 with an excitation filter of 330385 nm and barrier filter of 420 nm. TE fluoresce red and ICM appear blue.
Statistical analysis
The data on development and survival rate (percentage) after cryopreservation were arcsin transformed. Data were submitted to one-way ANOVA and Duncans test for means, and expressed as mean ± SEM (development, cryopreservation, and cell numbers).
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Results |
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Interestingly, the presence of RA led to oocytes exhibiting CG migration at the end of pre-maturation (group 4). In addition, oocytes from group 8 showed less clustering than the other groups analysed, and their CG formed a uniform monolayer beneath the oolemma (pattern 4). This monolayer, seen in six out of seven oocytes analysed, was absent in other groups, which suggests that the presence of RA during pre-maturation improves granular migration. Pictures from metaphase 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 blastocysts from oocytes treated with RA during roscovitine-induced pre-maturation but not during subsequent maturation, were comparable with those from uninhibited oocytes whereas the remaining groups developed at lower rates. Proportions of embryos at the 58-cell stage (not shown in Tables) were higher for uninhibited oocytes (64.9 ± 4.1) than for their prematured counterparts (values comprised between 42.9 ± 5.1 and 45.3 ± 7.7; P < 0.05). Treatment with RA during pre-maturation or maturation alone increased blastocyst development rates at 4 h post-warming (Table III
). However, blastocysts derived from a pre-maturation treatment with RA survived longer as compared with other groups and showed improved hatching. Survival differences observed at 72 h were the same as at 48 h. In Table IV
, oocytes treated with RA at any time tended to give blastocysts having higher ICM counts, and RA during pre-maturation alone increased total cell numbers. Embryos derived from oocytes exposed to RA during pre-maturation and maturation showed a striking reduction in TE cell numbers, which demonstrates an abnormal cell distribution.
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Discussion |
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Pre-maturation in the presence of RA improves cytoplasmic competence of in-vitro matured bovine oocytes from COC. Upon collection, CG are distributed in the cytoplasm of oocytes at the GV stage. As maturation proceeds, the granules migrate to the cortex and occupy the area just beneath the oolemma, at same time the nucleus enters MII stage. Cortical granule migration is a common phenomenon in mammalian oocytes during maturation both in vivo and in vitro (Yanagimachi, 1994; Wang et al., 1997a
). This migration is associated with a gain in developmental competence by the oocyte (Hosoe and Shioya, 1997
) and blocks polyspermy once migrated CG contents are released (Hyttel et al., 1988
; Wang et al., 1997b
; Nagano et al., 1999
). The most relevant finding within our CG migration study was probably that RA induced CG migration prior to maturation. Also, the CG distribution after RA exposure formed a uniform monolayer beneath the oolemma with lesser clustering once RA-prematured oocytes were allowed to mature in the absence of RA. Taken together, these results suggest a role for RA in the improvement of developmental competence. However, the exact timing (and possibly also the concentration) of RA exposure is critical since it alters the normal CG migration and distribution. The area devoid of granules immediately overlying the second meiotic spindle (Szollosi, 1967
; Nicosia et al., 1977
) has been called the CG-free domain (CGFD) (Ducibella et al., 1988
). Development of the CGFD in hamster and mouse oocytes is a result of local exocytosis and redistribution of CG (Okada et al., 1986
; Ducibella et al., 1988
). However, as previously described in the bovine (Long et al., 1994
), no obvious CGFD was observed in oocytes in the current experiment during meiotic maturation. Similar to observations in the mouse (Szollosi, 1967
) we did not observe migration of CG to the deeper cytoplasmic portion in the bovine. As reported previously, bovine CG organization becomes discontinuous in comparison with horse oocytes (Wang et al., 1997b
).
Embryo quality may be assessed by the number of embryonic cells (Ellington et al., 1990), and in-vitro produced blastocysts are often selected for freezing or vitrification according to quality. In-vitro re-expansion and hatching using somatic cell co-culture after cryopreservation is a common test for embryonic viability. In the present work, day 7 embryos belonging to groups having the highest total cell numbers on day 8 survived better to post-warming using co-culture. However, ICM cells and the ratio ICM/trophectoderm cells varied without affecting in-vitro survival and hatching, which has been described elsewhere (Thompson et al., 1995
; Van Soom et al., 1996
). The best criterion for viability is the birth of normal calves, which does not always equate with high embryo survival rates after thawing (Van Wagtendonk-de Leeuw et al., 1995; Donnay et al., 1998
). In the present work, RA exerted a proliferative effect which was not evident during maturation of bovine oocytes exposed to meiotic arrest, regardless of whether RA was present during pre-maturation or not. Furthermore, prolonged exposure of COC to RA caused embryos to have an abnormally low total cell number, a disproportionate embryonic cell distribution and reduced ability to survive cryopreservation, which could be interpreted as a low embryonic viability. The reduction in cell numbers only affected trophoblast cells, the ICM having a cell number range comparable to normal. Interestingly, the combined effects of pre-maturation together with the presence of RA during the pre-maturation period only increased trophectoderm cell counts and, as a consequence, total cell numbers in blastocysts. In addition, the number of cells allocated to the ICM had a tendency to increase in those blastocysts derived from oocytes treated with RA, in accordance with previous observations (Duque et al., 2002
) on blastocyst development and survival after vitrification and warming. Our results are not inconsistent with previous data showing that the presence of RA during non-inhibited bovine IVM improves blastocyst development (S.Ikeda, personal communication; Duque et al., 2002
) and increases numbers and the proportions of cells allocated to the ICM (Duque et al., 2002
). The influence of RA on GV-arrested oocytes could be correlated to weak [3H]uridine labelling of the GV area (Pavlok et al., 2000
) together with incomplete inactivation of hnRNA synthesis (Hyttel et al., 1997
) at this stage. RA may thus contribute to direct or indirect epigenetic processes influencing developmental capacity.
The detrimental effect of a RA overexposure (i.e. the presence of RA during both pre-maturation and maturation), or the neutral effect observed during maturation alone, may rely on an interaction of RA with FSH. As a consequence, RA diminishes FSH-induced expression of LH receptor in porcine (Hattori et al., 2000) and rat (Minegishi et al., 2000
) granulosa cells. Nevertheless, when porcine granulosa cells are treated with RA and washed before addition of FSH and LH, the expression of LH receptor mRNA is not reversed (Hattori et al., 2000
). Following the proposed model, treating COC with RA during pre-maturation alone would permit the expression of LH receptor within the cumulus cells. Although in our work concomitant FSH and RA was not beneficial, neither was a deleterious effect was observed.
Our preliminary observations suggests an involvement of midkine in RA effects. This influence is currently quantitatively investigated. Midkine has been isolated in bovine follicular fluid (Ohyama et al., 1994), and in rat granulosa cells is dependent on gonadotrophins (Karino et al., 1995
; Minegishi et al., 1996
). Since simultaneous addition of FSH and RA to a granulosa cell culture did not significantly change the accumulation of midkine mRNA observed with either FSH or RA alone (Minegishi et al., 1996
), this effect could be associated with the suppression of FSH receptor by RA (Minegishi et al., 1996
, 2000
). Recombinant midkine enhanced development to the blastocyst stage of bovine oocytes, its effect being mainly mediated by cumulusgranulosa cells during in-vitro maturation (Ikeda et al., 2000a
,b
). The need to ensure the supply of retinoids to the embryos within a physiological range makes the choice of dosage for retinoids a delicate matter. As improved blastocyst development and quality was obtained with 5 nmol/l RA under permissive maturation conditions (Duque et al., 2002
), we adopted this concentration in our experiments. Some other reports of the effects of RA on granulosa cells were also considered. Among them RA resulted in a 2-fold increase in midkine mRNA levels (0.3 µmol/l) (Minegishi et al., 1996
) and altered LH receptor mRNA expression (1 µmol/l) (Hattori et al., 2000
). The retinol concentration in human serum is 12 µmol/l, and fetal 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 isomere has been reported to be 25 times less potent than 9-cis-RA (Thaller et al., 1993
). In the present study a marked response was obtained by overexposure of bovine oocytes to retinoids. These detrimental effects could be related to a well documented retinoid imbalance associated with developmental abnormality (Morriss-Kay and Ward, 1999). Again, midkine could be an intermediary in these developmental phenomena (Griffith and Zile, 2000
). In fact, both excess and deficiency of retinoids cause abundant teratogenic defects due to its pleiotrophic activity. An analogous detrimental effect has been suggested with oocytes matured with RA recovered from cows treated with 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 to granulosa cells from mature follicles and luteal cells (Bucco et al., 1995; Wardlaw et al., 1997
; Zheng et al., 1999
), but not found in other species. CRABP control the access of RA to the cell nucleus protecting against the RA excess. The late intrafollicular expression of CRABP II could mean that this period is more sensitive to RA and needs protection. However, immature bovine oocyte and granulosa cells might show a better tolerance to, and/or a higher dependence on, RA, as they lack a system to regulate the retinoid levels. This reinforces the need of keeping RA within physiological levels in vitro, to perform RA doseresponse studies and to investigate the expression of retinoid binding proteins in the bovine follicle.
Until more is known about the molecular processes, targets and regulation of RA in the follicle and oocyte in experimental systems such as those presented in this study, it appears premature and irresponsible to use RA in maturation of human oocytes, especially in view of the narrow time window and possible species-specific differences in susceptibility and protection ot the oocyte from epigenetic influences of retinol. This is important with respect to the potentially teratogenic and irreversible long-term effects of RA resulting in congenital abnormalities during embryogenesis and unknown alterations in gene expression even later in life.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 2, 2002; resubmitted on March 14, 2002; accepted on May 24, 2002.