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RESEARCH |
Area de Selección y Reproducción Animal (SERIDA) and 1 Area de Genética y Reproducción Animal (SERIDA), Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Camino de los Claveles, 604 – Somio, Gijón 33203, Asturias, Spain, 2 Cooperativa de Agricultores de Gijón, Carretera Carbonera s/n Gijón, Spain and 3 Consejería de Medio Rural y Pesca, Navia, Asturias, Spain
Correspondence should be addressed to E Gómez; Email: egomez{at}serida.org
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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In the live cell, biologically active retinoids mediate their effects by binding to two different kinds of nuclear receptors, namely, RA receptors (RARs) and retinoid X receptors (RXRs), each having three subtypes, namely
, ß and 
. Upon entering the nucleus all-trans RA and 9-cis RA can activate RARs, whereas RXRs are activated only by 9-cis-RA (Mangelsdorf et al. 1994, Chambon 1996). Heterodimerization between RXR and RAR or other members of the steroid/thyroid hormone nuclear receptor superfamily (Chambon 1996), can occur in response to ligand-mediated activation. Heterodimers are capable of binding to specific DNA sequences named RA response elements, to increase or decrease the expression of a gene. Several component of the RA signalling pathway such as RAR and , RXR and ß, retinaldehyde dehydrogenase and peroxisome-proliferator activated receptor gamma, are expressed in bovine oocytes and embryos (Mohan et al. 2001, 2002) and in cumulus cells (Mohan et al. 2003). Immunostaining detected RAR, and RARß in blastocysts (Mohan et al. 2001, 2002) and RAR and RXRß in cumulus cells (Mohan et al. 2003).
It has been reported that the developmental competence of the oocyte is enhanced by ROH treatment in the cow (Shaw et al. 1995), sheep (Eberhardt et al. 1999) and gilt (Whaley et al. 1997, 2000), as well as in rabbits that have high blood levels of vitamin A (Besenfelder et al. 1996). In studies involving cows (Shaw et al. 1995), sheep (Eberhardt et al. 1999) and gilts (Whaley et al. 1997, 2000) the effects of retinol on follicular development were monitored and recorded 4 days post-administration. The reason why these standards were set are not described and the limits (length and dosage) of ROH treatment are unknown. Furthermore, it would be interesting to determine whether the effects of exogenous ROH were exerted on the intrafollicular oocyte, the ovulated oocyte, the proper early embryo, or both the oocyte and the embryo.
The ability of blastocysts resulting from oocytes of domestic females treated with exogenous ROH to establish and maintain pregnancy is unknown. Development of these blastocysts after transfer to recipients is worth testing since disturbances in retinoid homeostasis have been linked to developmental abnormalities (reviewed by Zile 2001). The above questions can be tested in part by treating oocyte donor cows with ROH, as those oocytes aspirated from the follicle would develop free from the effects of circulating ROH when cultured in vitro up to the blastocyst stage. We have earlier reported that the presence of RA in in vitro maturation (IVM) medium enhanced oocyte maturation, embryo development (Duque et al. 2002a, Gómez et al. 2003, 2004, Hidalgo et al. 2003) and pregnancy rates (Hidalgo et al. 2003). Since ROH has no effect during IVM at physiological temperature (Duque et al. 2002b, Lawrence et al. 2004) it was necessary to perform an overexposure of the oocytes to retinoid by using RA. Therefore, our study is intended to analyse whether ROH acts in the cow as early as the oocyte intrafollicular growth period, to contribute to define limits and timing of the oocyte exposure to exogenous ROH, and to determine the efficiency and sustainability of Ovum-Pick Up (OPU) techniques based on ROH support in terms of oocyte and blastocyst yields and viability of embryos upon transfer to recipients.
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Materials and Methods |
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Animal management
Asturiana de los Valles heifers aged 17–20 months were fed freely with home-made grass silage, mineral premix, and barley or oat straw along with 1–2 kg concentrate per day in order to maintain a body condition score of 2.5–3 (scale 1–5, where 1 emaciated and 5 obese). All experimental animals were housed, handled and used in accordance with Spanish national laws. Prior to an OPU session, each animal was restrained in a standing position in a chute and tranquillized with 2% xilacine (0.25 ml/kg live weight of Rompun; Bayer, Barcelona, Spain). Rectum and perineal regions were cleaned and disinfected prior to administering anaesthesia. Epidural anaesthesia was performed using 2% lidocaine (Laboratorios Ovejero, León, Spain). Subsequent to oocyte aspiration each donor cow received a vaginal wash with povidone-iodine (10% Betadine).
Follicle puncture
To recover oocytes by OPU, ovarian follicles were visualized with a 6.5 MHz convex echographic transducer (PB-06VE65/20BD; Sonovet, Kangwon-do, Korea) connected in parallel to an echo camera/ultrasound scanner SA 600 (Sonovet, Kangwon-do, Korea). The probe incorporated a guide through which a disposable puncture needle was introduced (V-OPAN-1760; Cook Veterinary Products, Queensland, Australia) and attached to Teflon tubing (internal and external diameter: 1.6 and 3.2 mm respectively). Tubing was connected to a 50 ml collection tube (Corning Inc., NY, USA) and the system was powered by a vacuum pump (V-MAR-5100; Cook Veterinary Products) providing pressure of 85 mmHg (equivalent to a water flow of 14 ml/min). The collection tube was kept at 37°C in a heater (V-FTH-2012; Cook Veterinary Products). Before performing the procedure the system was rinsed with collection medium (CM) (TCM199; Invitrogen) with 25 mM Hepes, 0.4 g/l BSA Fraction V and 10 IU/ml heparin). The flow was periodically monitored and the system was rinsed with CM after the end of the session.
Collection of follicular fluid (FF) and blood, and retinol analysis
FF aspirated from single follicles were transferred into 1.5 ml Eppendorf tubes. After measuring the aspirate volume the tubes were centrifuged and the supernatant was frozen at –80 °C until further analysis. Following each collection, the aspiration circuit was extensively rinsed with physiological saline.
Blood samples were collected from coccygeal veins using vacutainer tubes containing no additives. Samples were centrifuged and the serum was stored at –80 °C until further analysis. ROH concentration was determined by a quality-controlled HPLC method (Olmedilla et al. 1992). In brief, variable amounts of serum and FF (50–500 µl) were thawed and deproteinized with ethanol containing an internal standard (retinyl acetate). After vortexing for 45 s, analytes were extracted twice with hexane/methylene chloride (5:1 v/v), and the organic phases pooled, evaporated and reconstituted before injection into the HPLC equipment. Chromatographic analysis was carried out on Spheri-5-ODS (5 µm, 4.6 x 250 mm) (Applied Biosystems, San José, CA, USA), and isocratic elution was performed with acetonitrile/methylene with acetonitrile/methylene chloride/methanol (ammonium acetate 0.025 M) (70:20:10) at a flow rate of 1.3 ml/min. Peak identification was confirmed by chromatographic behaviour and on-line spectra with a 996 Photodiode Array Detector (Waters Associates), compared with those obtained for standards and registered in the Millennium Data Station (Waters Associates). Retinyl acetate and ROH were detected at 325 nm. Using this protocol our quantification limit was 0.5 µg/dl.
Accuracy and precision of the analytical method used was evaluated periodically through participation in the Fat-Soluble Vitamins Quality Assurance Program conducted by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). Accuracy was within 10% from assigned values, and within-day and between-day precision for evaluated analytes during the period of this study, assessed by blind duplicates distributed by NIST, were <5% and <10% respectively.
Oocyte search and classification
FF contents form each heifer were filtered (Emcon; Comex-trade, Tarragona, Spain), rinsed with ovum culture medium + 5% fetal calf serum (FCS) and placed in a Petri dish. Intact cumulus-oocyte complexes (COCs) were picked using a stereomicroscope and washed four times in holding medium (HM) (TCM199 + 25 mM Hepes +0.4 g/l BSA). COCs were then classified into four categories based on their morphological appearance at x 60: (1) complete and compact cumulus, having at least four complete layers of clear cells; (2) complete but less compact cumulus, with at least four cell layers, or abnormally dark cells; (3) incomplete or expanded cumulus; and (4) denuded oocytes. Degenerated and broken oocytes were recorded but excluded from culture.
In vitro embryo production
For IVM, we used a basic maturation medium (MM) consisting of TCM199 + NaHCO3 (2.2 g/l) + porcine follicle-stimulating hormone (FSH) (1 µg/ml) + luteinizing hormone (LH) (5 µg/ml) + 17ß-oestradiol (1 µg/ml) + poly vinyl alcohol (PVA) (0.5 mg/ml). After two washes in MM and a new wash in the medium assigned to each group, COCs were matured in 500 µl MM in four-well dishes (Nunc, Roskilde, Denmark) for 22–24 h at 39 °C, in an atmosphere containing 5% CO2 and saturated humidity. All categories of COCs described were used for IVM.
For in vitro fertilization (IVF), motile spermatozoa were separated by a swim-up procedure similar to that reported by Parrish et al.(1986). Frozen–thawed semen was layered into 1 ml sperm Tyrode–albumin–lactate–pyruvate (TALP) and incubated for 1 h. The upper 700 ml containing motile spermatozoa was centrifuged for 7 min at 100 g and the supernatant was aspirated leaving a pellet of approximately 100 µl in volume. Spermatozoa concentration was then determined using a haemocytometer. The COCs were washed twice in HM, twice in pre-equilibrated fertilization medium (Fert-TALP), and placed in four-well culture dishes containing Fert-TALP with heparin (10 µg/ml) (Calbiochem, La Jolla, CA, USA). Spermatozoa were then added at a concentration of 2 x 106 cells/ml in 500 µl of medium per well. IVF was accomplished by incubating oocytes and spermatozoa together for 18–20 h at 39 °C in 5% CO2 under high humidity.
For in vitro embryo culture, presumptive zygotes were gently passed back and forth through a small-gauge pipette to remove cumulus cells and sperm, washed three times in HM and twice in culture medium. Embryo culture was performed in modified synthetic oviduct fluid (285 mOsm, pH 7.2–7.3) containing amino acids, citrate and myo-inositol (Holm et al. 1999). FCS was added to the embryo culture media 42 h post-fertilization (PI). Droplets of culture medium (1–2 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.
Embryo transfer (ET)
Day 7 blastocysts were selected for ET. Embryos transferred fresh included unexpanded and expanded blastocysts, while embryos transferred after vitrification and warming were predominantly expanded blastocysts. For vitrification, embryos were loaded into straws containing PBS (Invitrogen) with glycerol, ethylene glycol and FCS, adjacent to two fractions of galactose solution (Kaidi et al. 2001). Embryos were warmed for 5 s in air and 10 s in a water bath at 30 °C. The contents of each straw were emptied into a Petri dish and embryos were mixed in the galactose solution by slight agitation. After 5 min, the embryos were transferred to another Petri dish containing PBS + 20% FCS for 5 min. Embryos were then allowed to re-expand in B2 INRA Ménézo (C.C.D., Paris, France) at 39 °C in an incubator. Finally, re-expanded embryos were loaded into 0.25 ml straws and non-surgically transferred into synchronized recipients, aged 15–18 months.
Experimental design
Experiment 1: ROH concentration in blood and FF after administration of ROH
We first studied short-term responses to a single ROH injection using 12 heifers. Oestrus was synchronized with progestagen (Crestar; Intervet, Salamanca, Spain) and then followed with an injection of prostaglandin F2 (Estrumate; Shering-Plough, Kenilworth, New Jersey, USA) given on Day –2 of the subsequent cycle. Blood samples were first taken at the time of progestagen removal (Day 0), just prior to treatment administration. Animals were then injected with 1 x 106 IU ROH (Fluka 95144; Madrid, Spain) (n = 6) or corn oil (vehicle; n = 6) on Day 0. Blood samples were taken again on Day +1 and Day +4, and contents of single follicles measuring between 3 and 10 mm in diameter were aspirated on Day +4, and the recovered FF volume was measured. Blood plasma and FF were analysed for ROH.
Experiment 2: Influence of exogenous ROH on oocyte production and development
This experiment was performed to investigate the effect of repeated OPU sessions following ROH administration. Oocyte donors (n = 26) were subjected to one cycle of eight OPU sessions without superovulation at alternating intervals of 3 and 4 days, as can be seen in Table 1
. Animals were injected with 1 x 106 IU ROH (n = 13) 4 days before the first session and at the end of the second, fourth and sixth sessions. Four days later, a second OPU cycle of eight sessions started without ROH stimulation. Control groups of heifers (n = 13) received corn oil (vehicle) during the first cycle and were not treated in the second cycle. Blood plasma was analysed for ROH twice per week during the first cycle and once per week during the second cycle. Visible follicles were counted and aspirated. Recovered COCs were counted and classified as mentioned earlier. COCs obtained from similarly treated donors were pooled and assigned to each of two IVM groups that contained either 0 or 0.5 µM 9-cis-RA in a 2 x 2 factorial design, according to ROH-treated donors. Variables monitored were follicle counts, oocytes recovered, oocyte quality and oocytes selected for IVM; cleaved zygotes and embryos with more than eight cells on Day 3; and blastocyst development on Day 7 and Day 8. Cows were managed in groups of four and six animals, and the experiment was repeated five times. Cultures with no embryonic development in all groups were not considered for analysis. Three donors could not be used beyond the fourth session in the first cycle, and two more donors during the entire second cycle because of clinical problems.
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Statistical analysis
Data from follicle numbers, oocyte production and embryo development were analysed in two steps. First, since these values can be represented by contingency tables, those factors significantly affecting the variables were identified by means of the procedure Categorical Data Modelling (CATMOD) (SAS Version 8.2 1999; SAS Institute Inc., Cary, NC, USA). CATMOD extends analysis from continuous data to categorical data, fitting linear models to functions of response frequencies. Treatment and replicate were found to have significant influence on dependent variables. Secondly, those factors identified to be significant were used to fit a linear model using the procedure General Linear Models (GLM) (SAS). This procedure performs ANOVA for unbalanced data. GLM was used to estimate least square means (LSM) for each fixed effect having a significant F value. Duncan’s multiple-range test was carried out on raw means calculated for the main effects on embryo development. LSMs for monitored follicles, oocyte production and embryonic development, are expressed as absolute average values on a per donor and per treatment basis, although embryonic development data were as well referred to frequency percentages of oocytes in IVM. Significant interactions between ROH and RA were detected by means of CATMOD. GLM was used to reject the null hypothesis (H0: LSM(i) = LSM(j)) for LSMs within interaction levels. Data for ROH concentrations were processed by GLM–ANOVA and Duncan’s test. Pregnancy data were submitted to chi-square test of homogeneity for each stratum of treatment-day of pregnancy, and results refer to absolute frequencies.
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Results |
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), and this rise was cleared 4 days after injection. On Day +4, one single follicle was aspirated per cow (n = 12) and, interestingly, ROH concentration was higher in follicles in cows treated with ROH compared with controls. Stage of the oestrous cycle did not influence blood and FF ROH concentrations. The average FF volume recovered was 393 ± 127 µl (range 80–950) and 351 ± 127 µl (range 100–700) for ROH and oil-treated heifers respectively (P > 0.8), and the measured volume of FF correlated with ROH concentrations (r = 0.70, P = 0.034).
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), the samples were recovered at the end of each session in the first cycle (twice per week) and after the first, third, fifth and seventh sessions within the second cycle (once per week). Exogenous administration of ROH gave no differences (P > 0.15) between and within the treated first cycle and the untreated second cycle (Table 2).
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. A gradual decrease in follicle numbers and oocytes recovered, although independent from treatment, was observed. Overall, the first cycle was more productive than the second cycle (Table 3), and ROH injected within the first cycle had no significant influence on subsequent oocyte production during the second cycle. More low-quality oocytes were harvested in the first cycle from donors treated with ROH.
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. While no differences could be observed between cleavage and the five to eight cell stage (not shown in Table), embryos matured with RA showed higher developmental kinetics on Day 3, as more embryos developed to the 8–16 cell stage. During the first cycle, RA increased the formation of morula but did not increase blastocyst formation rates. However, cumulatively, RA did increase blastocyst rates.
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). Anticipating overall results, COCs yields within the ROH group accounted for higher numbers of low-quality recovered COCs (categories 3 and 4), as well as for higher follicle numbers. Interestingly, more morulae and blastocysts developed from oocytes collected during sessions one to five from ROH-treated animals (Table 6). On the other hand, embryonic development observed with oocytes obtained from control animals treated with RA was even better than those observed with ROH-treated animals. Differences were not observed later than session five since oocytes recovered in subsequent sessions during both the first and second cycle did not respond to RA.
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). As shown by low P4 levels on Day 21, development of transferred blastocysts was most probably disrupted before implantation. Pregnancy rates were independent of whether fresh or vitrified embryos were transferred.
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Discussion |
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Gonadotrophins such as FSH either alone (Bousquet et al. 1999, Goodhand et al. 1999, Sirard et al. 1999) or in combination with LH (Blondin et al. 2002, see a review by Merton et al. 2003) and pregnant mare serum gonadotrophin (Pieterse et al. 1991) have been used to improve the outcome of OPU in adult donor cows. Oocytes from superovulated cows are developmentally more competent than their non-stimulated counterparts (Faber et al. 2003, Merton et al. 2003), and specific changes in superovulatory treatment can greatly increase developmental competence of the oocyte (Blondin et al. 2002). However, due to high variability among gonadotrophin batches and being an animal product we considered ROH as a potential alternative or complementary treatment to hormonal stimulation. In addition, OPU performed at 3- or 4-day intervals is a scheme that can be used for up to 4 consecutive months (Petyim et al. 2003) and has been shown to yield more oocytes per session and embryos from each oocyte batch collected at 3-day intervals (Merton et al. 2003), although we found no significant differences between same intervals (data non shown). Our hypothesis is further supported by the fact that retinoid treatment has been previously shown to increase litter size in the gilt (Brief & Chew 1983, Coffey & Britt 1993) and rabbit (Besenfelder et al. 1996), and improve embryonic quality without modifying ovulation rates (Shaw et al. 1995) in superovulated cows (Shaw et al. 1995) and ewes (Eberhardt et al. 1999).
During the first oocyte recovery sessions ROH increased the total number of monitored follicles and aspirated oocytes. However, most of the oocytes recovered were of low quality. In the present study we found that oocyte donors were more responsive to ROH following the first three doses, giving us some indication regarding the optimal length of ROH treatment. Oocytes from cows treated with ROH showed reduced morulae and blastocysts formation rates, analogous to that seen in an earlier study with oocytes continuously stimulated with RA during IVM in the presence of a meiotic inhibitor (Duque et al. 2002). It has been described that resistance to RA, a phenomenon involving a multifactorial aetiology that is not very well understood, develops rapidly (Marill et al. 2003) and could be involved in decreased late responses observed in our cows and oocytes. Higher transition rates by the eight cell stage were observed with in vivo embryos from ewes treated with ROH (Eberhardt et al. 1999), but in our study we did not observe this effect with ROH. Nevertheless, the ROH metabolite RA gave more embryos produced by OPU at the 8–16 cell stage on Day 3. This effect was not observed in our previous experiments with slaughterhouse oocytes, so it probably depends on heterogeneous quality within cultured oocytes and/or the OPU procedure itself. The oocyte transcriptional machinery and protein synthesis can be altered by RA stimulation through activating specific genes such as midkine (Gómez et al. 2003). The recombinant peptide product of midkine exerts beneficial effects on embryonic development when added during IVM (Ikeda et al. 2000a,b), although it is also an intermediary candidate in the induction of developmental alterations (Griffith & Zile 2000). The stimulation by RA at IVM triggers a more physiological cytoplasmic granule migration (Duque et al. 2002) and increases oocyte poly-adenylation, suggesting superior mRNA quality and increased processing (Gómez et al. 2004). This coincides with unaltered expression levels of cyclins B1 and H1, specific genes regulating the cell cycle, and less oxidative damage (Gómez et al. 2004). Altogether, the above findings correspond to more developmentally competent oocytes (Duque et al. 2002, Hidalgo et al. 2003). The ovarian response to FSH and LH can also be modulated by RA. In response to this retinoid, stimulation of steroidogenesis (Bugavandoss & Midgley 1988, Graves-Hoagland et al. 1988), and restricted expression of FSH receptor (FSHr) in rat granulosa cells has been reported (Minegishi et al. 1996, 2000), which could affect oocyte growth and in turn the outcome of OPU in the cow (Argov et al. 2004). In the porcine, the effects are controversial, as exposure of immature granulosa cells to RA inhibited expression of LH receptor (LHr) by downregulation of c-Fos mRNA (Hattori et al. 2000). In addition, feeding gilts with ROH did not affect P4 secretion by granulosa cells with or without LH stimulation (Whaley et al. 2000). In the bovine, decreased levels of FSHr and LHr in cumulus-granulosa cells have been shown to be associated with low-quality oocytes (Calder et al. 2003). In the presence of RA, the growth hormone (GH) receptor (GHr) gene increases transcription (Ohlsson et al. 1993, Slootweg et al. 1996). The transcript of the GHr was demonstrated in oocytes, follicular cells and the corpus luteum of the bovine ovary (Kolle et al. 1998), and GH was found to stimulate the bovine oocyte during IVM via cumulus cells (Izadyar et al. 1997, Kolle et al. 2003).
In the present study, development of blastocysts from cows that received multiple ROH injections was disrupted before implantation. This is in contrast to pregnancies obtained when RA was present during IVM (Hidalgo et al. 2003), as these embryos were able to survive beyond the expected time for completion of organogenesis (Day 42) (Hubbert 1974). Vitamin A deficiency and excess can adversely affect the development of the vertebrate embryo as almost every organ or tissue can be affected if there is an imbalance in RA homeostasis during certain critical stages of development (reviewed by Zile 2001). Adequate metabolic systems must inactivate unused RA in order to maintain a stringent retinoid balance, as additional RA synthesis by the embryo is essential for post-implantation development (Niederreither et al. 1999). Most studies on retinoids have focused on later stages of embryonic development, although exposure of zygotes to RA during early embryonic development yields anomalies during pre-gastrulation and organogenesis (Rutledge 1997). Recently, RA treatment during the pre-implantation period in the mouse (Huang et al. 2001, 2003) was shown to cause developmental retardation, inhibition of cell proliferation and induction of apoptosis. In previous reports, development of blastocysts to term was not analysed (Shaw et al. 1995, Whaley et al. 1997, 2000, Eberhardt et al. 1999), and the precise stage (oocyte, embryo or both) during which retinoids exerted their effects was not determined. Our results involving oocytes isolated by OPU following ROH treatment and subsequently cultured in vitro demonstrate that ROH can influence development by acting on the oocyte during its intrafollicular growth. In the future, it would be interesting to find out the outcome had the oocyte following ROH treatment been fertilized in vivo and the embryo recovered at specific stages during pre-implantation development, as done in earlier studies (Shaw et al. 1995, Besenfelder et al. 1996, Whaley et al. 1997, 2000, Eberhardt et al. 1999).
It is interesting to note that in the present study, the development of embryos following transfer was negatively influenced by ROH treatment of the oocyte donor cow while RA treatment of the oocyte during IVM in our earlier studies promoted oocyte developmental competence. The animal cell contains all the components required to bind and regulate the release of retinoids, from ß-carotene up to the nuclear effector RA. It is possible that this system could have failed since repeated ROH administration did not produce long-term increases in blood ROH concentrations above physiological levels. Instead, excess ROH could have been compartmentalized and stored (i.e. the follicle). Serum-derived follicular ROH can bind to specific protein in granulosa cells (Brown et al. 2003) and the oocyte (Mohan et al. 2001). After entry into the cell, ROH can be metabolized to RA, which can then be bound by specific CRABPs. As previously demonstrated in rats (Bucco et al. 1995, Wardlaw et al. 1997, Zheng et al. 1999), intracellular proteins that regulate RA availability may not be expressed in immature follicles; as a result the availability of RA in the follicle could have gone unchecked. On the contrary, under in vitro conditions, by providing a carefully calculated dose, it is possible to ensure that an optimum concentration of RA is made available to the COCs during in vitro culture.
The present study confirms the existence of an effect of ROH on the bovine oocyte during intrafollicular growth. As a single dose, injected ROH increases ROH concentration in blood by 24 h. By 4 days after injection, blood ROH clearance was observed, but interestingly ROH remained higher within the follicle. Weekly repeated ROH injections enabled the recovery of higher numbers of low-quality oocytes by OPU throughout the cycles studied. Furthermore, total oocytes recovered as well as morulae and blastocyst formation rates increased during the first five sessions in cows treated with ROH. However, ROH levels measured during OPU cycles did not change, suggesting the non-existence of ROH downregulation. On the other hand, as previously demonstrated using oocytes from slaughterhouse ovaries, RA stimulated blastocyst development (Duque et al. 2002, Hidalgo et al. 2003). Accordingly, under the RA influence, higher proportions of embryos overcame the developmental block on Day 3 of culture. Once transferred to recipients, blastocysts derived from oocytes from cows treated with ROH were unable to set pregnancy, showing an early sign of teratogenic effect of ROH. Exposure to ROH and RA should be avoided in candidate mothers as early as possible prior to the fertilization time.
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Footnotes |
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Received 27 October 2004
First decision 26 November 2004
Revised manuscript received 10 December 2004
Accepted 17 December 2004
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References |
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) or vehicle (
). (b) Oocytes recovered during two cycles (retinol stimulated and unstimulated) in donor cows treated with retinol (