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Gamete Biology |
a Laboratories of Animal Reproduction
b Animal Genetics, Graduate School of Biosphere Science, and
c Laboratory of Animal Science, Graduate School of International Development and Cooperation, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, embryo, in vitro fertilization, luteinizing hormone, meiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes for in vitro maturation (IVM) are usually collected from early antral follicles (3–5 mm) of prepubertal gilts. When porcine cumulus oocyte complexes (COCs) from the antral follicles were cultured with LH, the level of cAMP in the cumulus cells surrounding oocytes was not increased [8], whereas FSH raised cAMP level in cumulus cells of mouse COCs [5, 11]. Reverse transcription polymerase chain reaction (RT-PCR) with cDNA of bovine cumulus cells or oocytes revealed that mRNA for FSH receptor was present in cumulus cells, however, mRNA for LHR was not detected in both cell types [6]. In mouse COCs, Chen et al. [12] reported that the low expression of LHR was also seen in cumulus cells immediately recovered from their follicles; however, the formation of the receptors was upregulated by FSH. These results indicate that LHR is expressed in cumulus cells of porcine COCs during in vitro meiotic maturation, and the binding of LH to the resultant receptors may be critical for in vitro and in vivo meiotic maturation of oocytes. However, Hattori et al. [13] reported that LHR mRNA in porcine cumulus cells was detected after 48 h of culture with FSH, whereas the LHR mRNA was absent from the putative transmembrane domain. Therefore, the investigations of time-dependent changes of LHR expression in cumulus cells and its function are required for a better understanding of in vitro meiotic maturation of porcine oocytes.
Several researchers have examined the effects of holding oocytes at the germinal vesicle (GV) stage before in vitro maturation, because oocytes might require time to acquire developmental competence during meiotic arrest [14–17]. Funahashi et al. [14] showed that exposure of COCs to dbcAMP for the early period of IVM synchronized the oocytes to the GV II stage and improved early embryonic development following in vitro fertilization (IVF). The results of our previous experiments [18] demonstrated a transient increase in the level of cAMP in porcine oocytes followed by a dramatic decrease in the level of cAMP before GV breakdown (GVBD). Additional phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), maintained a high level of cAMP and arrested meiosis at the GV II stage when porcine COCs were cultured with gonadotropin [18]. Thus, we hypothesized that holding oocytes at the GV II stage in medium containing FSH and IBMX would allow the cumulus cells to express LHR and the oocytes to develop to the proper GV stage for LH stimulation and might allow the oocytes to acquire greater developmental competence.
We investigated the dynamics in the formation of LHR in cumulus cells surrounding porcine oocytes and the role of LHR during meiotic maturation of oocytes. After COCs were cultured for 20 h in the FSH medium with or without IBMX, the expression of LHR mRNA was analyzed with an RT-PCR technique, and the level of LHR on a cell surface was detected using biotinylated hCG for a probe ligand. Furthermore, time course of nuclear maturation and subsequent developmental potential of oocytes cultured with LH following initial cultivation of COCs with FSH and IBMX were examined. We also investigated whether the binding of LH to its newly synthesized receptor on cumulus cells produced an increase in cAMP and progesterone production in COCs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of porcine COCs was described previously [19]. Porcine ovaries were collected from 5- to 7-mo-old prepubertal gilts at a local slaughterhouse. Oocytes were collected with a surgical blade from the surfaces of intact healthy antral follicles 3–5 mm in diameter. Oocytes having evenly granulated cytoplasm with at least four layers of unexpanded cumulus oophorous cells were selected. These oocytes were washed three times with maturation medium, i.e., modified NCSU37 [20] consisting of 10% (v/v) fetal calf serum (Gibco BRL, Grand Island, NY) and 7 mM taurine (Sigma, St. Louis, MO).
Twenty COCs were cultured in each well of a Nunc four-well multidish (Nunc, Roskilde, Denmark) containing 500 µl of culture medium at 39°C in a humidified atmosphere of 5% CO2 in air. Control experiments demonstrated that the osmolarity of cultures maintained under these conditions varied by <1% after 44 h.
Assessment of Nuclear Maturation
At the end of cultivation, COCs were freed from cumulus cells, mounted on slides, fixed with acetic acid/ethanol (1:3) for 48 h, and stained with acetolacmoid before examination under a phase-contrast microscope (400x) for evaluation of chromatin configuration.
RNA Isolation
After cumulus cells were separated from 20 COCs, they were washed three times in PBS. Total RNA was extracted from cumulus cells using the SV Total RNA Isolation System (Promega, Madison, WI), according to the instruction manual, and dissolved in 20 µl nuclease-free water.
RT-PCR
Oligonucleotide primers used for amplification of the LHR were designed from known cDNA sequences of four porcine LHR isoforms [4]. The upstream primer (5'-CCAATCTCCTAGATGCCACATTGAC-3') is identical to nucleotides 861–885 of the porcine cDNA, and the downstream primer (5'-GCTCAGCAACAGAAAGAAATCCC-3') represents the reverse complement of nucleotides 1959–1981. This primer pair predicts 185-, 411-, 855-, and 1121-base pair (bp) DNA fragments.
ß-Actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. The ß-actin primers were based on the mouse sequence [21]. The upstream primer (5'-CTACAATGAGCTGCGTGTGG-3') is identical to nucleotides 192–211 of the mouse cDNA, and the downstream primer (5'-TAGCTCTTCTCCAGGGAGGA-3') represents the reverse complement of nucleotides 622–641. The primer pair predicts a 450-bp DNA fragment.
RT-PCR was performed according to a coupled one-step procedure using the Access RT-PCR System (Promega). Total RNA was reverse transcribed at 48°C for 45 min, denatured at 94°C for 2 min, and amplified for 35 cycles of denaturation at 94°C for 30 sec, primer annealing at 60°C (LHR) or 58°C (ß-actin) for 1 min, and extension at 68°C for 2 min, with a final extension step of 7 min at 68°C. The amplified products were analyzed by electrophoresis on 2% agarose gels.
Biotinylation of hCG
Sulfo-NHS-LC-Biotin (2 mM; Pierce, Rockford, IL) was incubated with 0.1 mM hCG (NIDDK, Torrance, CA) for 2 h in PBS at 4°C. The endoproducts were separated from unbound Sulfo-NHS-LC-Biotin using a microconcentrator (UltraFree-C3-LGC; Millipore, Mississauga, ON, Canada). The biotinylated hCG was stored at 4°C in 0.1% (w/v) sodium azide. The degree of biotinylation of hCG was determined with the 2-(4'-hydroxyazobenzene) benzoic acid (Sigma) reaction according to the manufacturer's instructions.
Binding of Biotinylated hCG to Cumulus Cells Surrounding Oocytes
Twenty COCs were incubated in the basic medium supplemented with 5 µg/ml biotinylated hCG at 39°C for 1 h. To determine specificity of LH binding receptors, COCs were incubated with increasing concentrations from 0.1 to 10 µg/ml of porcine LH (NIDDK) for 1 h and then further incubated with 5 µg/ml of biotinylated hCG. After incubation with biotinylated hCG for 1 h, the COCs were washed three times with the basic medium. After washing, the COCs were further cultured in the basic medium supplemented with 0.67 µg/ml of horseradish peroxidase-conjugated avidin (Sigma) for 30 min at 39°C. The COCs were then washed five times with ice-cold PBS. These COCs were put into the plastic tubes containing 0.5 mM Tris-HCl (pH 7.4) and were broken down by repeated aspiration. Peroxidase activity was determined at 25°C using o-phenylenediamine (MBL, Nagoya, Japan) in 0.1 mM phosphate buffer (pH 5.1) and 0.015% (v/v) H2O2. After the addition of 20% (v/v) H2PO4 solution, the absorbance at 492 nm was measured on a spectrophotometer.
This assay is based on the ability of biotinylated hCG and LH to compete for binding to LHR in porcine COCs. With this method, the rate of nonspecific binding of biotinylated hCG to COCs was 4.1%.
Quantification of cAMP by HPLC-Ultraviolet Analysis
Quantification of cAMP by HPLC-ultraviolet (UV) was based on the procedures reported previously [18]. COC extracts were separated using a reverse-phase Eicompak CA-5DS column (2.1 x 150 mm; Eicom, Kyoto, Japan). The solvent delivery system (DP 8020; TOSOH, Tokyo, Japan) contained 97.2% (v/v) 0.01 M ammonium acetate (Nakalai) and 2.8% (v/v) acetonitrile (Nakalai), pH 6.7. The detection was performed at 254 nm using a UV detector (UV 8020; TOSOH), and peak heights were measured using a computer integrator (Sic chromatocorder 11; TOSOH).
Cyclic AMP (Sigma) was diluted in the assay buffer (100 µM) and kept frozen at -80°C. The standard solution was diluted in the assay buffer prior to analysis. The standard curve of cAMP for the determination of concentration was linear from 0 to <2 pmol. The intra-assay coefficient of variation (CV) in medium with 0.1 pmol of cAMP was 3.86%.
Quantification of Progesterone in Medium by HPLC-UV Analysis
Quantification of progesterone by HPLC-UV was based on the procedures reported previously [22]. The medium in which COCs had been cultured was collected into plastic tubes and centrifuged at 10 000 x g for 20 min. Progesterone was extracted from the medium by 5-min mixing with 10 ml dichloromethane (Nakalai). After centrifugation, the 10 ml of dichloromethane fraction was collected into a disposal tube, and the solvent from this fraction was removed by vacuum extraction for 120 min at 5°C. Samples were reconstituted in 100 µl of 50% (v/v) methanol solution.
The samples were separated using a reverse-phase CAPCELL PAK column (2.0 x 100 mm; Shiseido, Tokyo, Japan). The solvent delivery system contained 50% (v/v) methanol solution. The detections of progesterone were performed at 240 nm using a UV detector and peak heights were measured using a computer integrator. The standard curve of progesterone for the determination of concentration was linear, from zero to 800 ng/ml. The intra-assay CV in medium with 100 ng/ml of progesterone was 4.15%.
In Vitro Fertilization
Cumulus cell-free oocytes were washed three times with the fertilization medium, i.e., modified Tris-buffered medium (mTBM) supplemented with 0.1% (w/v) BSA (fraction V, A 7888; Sigma) and 1 mM caffeine (Sigma). After washing, 20 oocytes were placed in 50-µl drops of the fertilization medium that had been covered with mineral oil in a 35- x 10-mm2 polystyrene culture dish (Falcon, Franklin Lakes, NJ). The dishes were kept in the incubator for about 30 min until spermatozoa were added for fertilization. Frozen epidermal spermatozoa [23] from a Gottingen Miniature Boar were thawed and washed by centrifugation at 700 x g for 5 min in washing medium, i.e., mTBM supplemented with 0.1% (w/v) BSA. The sperm pellet was resuspended and precultured for 90 min in precultured medium, i.e., mTBM supplemented with 10% (v/v) fetal calf serum and 1 mM caffeine. The concentration of spermatozoa during precultivation was 2 x 108 cells/ml. The precultured spermatozoa were diluted to 2 x 106 cells/ml in the fertilization medium, and 50 µl of this sperm suspension was added to 50 µl of the fertilization medium that contained oocytes (final concentration of sperm, 106 cells/ml). Oocytes were cocultured with spermatozoa at 39°C in an atmosphere of 5% CO2 in air for 6 h. The mTBM used for IVF was essentially the same as that used by Abeydeera and Day [24]. Because the pH of basic mTBM just after preparation is about 9.8–10, final IVF medium was kept in the incubator (an atmosphere of 5% CO2 in air at 39°C) for 18–24 h to stabilize the pH to 7.2–7.3 before use.
In Vitro Production of Embryos
After sperm-oocyte coincubation, putative zygotes were cultured as described by Kikuchi et al. [25]. After coincubation of the gametes for 6 h, the putative zygotes were washed three times and transferred into in vitro culture (IVC) medium. The day of insemination was defined as Day 0. The basic IVC medium was NCSU 37 medium containing 0.4% BSA (fraction V, A 8022; Sigma). In vitro production (IVP) embryos were cultured in IVC medium supplemented with 0.17 mM sodium pyruvate (Sigma) and 2.73 mM sodium lactate from Day 0 to Day 2, then in IVC medium with 5.55 mM D-glucose, as previously described [20], from Day 2 to Day 6. At 12 and 144 h after IVF, the proportions of pronuclear formation and blastocyst formation, respectively, were evaluated.
Embryo Evaluation
To examine their ability to develop to the blastocyst stage in vitro, all embryos were cultured for 6 days, fixed with acetic acid:ethanol (1:3) for 48 h, and stained with acetolacmoid. An embryo with a clear blastocele was defined as a blastocyst for the purposes of this study. The rate of blastocyst formation was evaluated, and the total number of cells in each blastocyst was determined as an indicator of embryo quality.
Statistical Analysis
Statistical analyses of all data from three or four replicates were carried out by one-way ANOVA followed by a Duncan multiple-range test (Statview; Abacus Concepts, Berkeley, CA). All percentage data were subjected to arcsine transformation before analysis. Differences were considered significant at P < 0.05.
Experimental Design
Experiment 1 was conducted to examine time-dependent changes of LHR formation in cumulus cells of COCs. Porcine COCs were cultured with 20 ng/ml porcine FSH (NIDDK) for 0, 8, 12, 16, or 20 h. After cultivation of COCs, the expression of LHR mRNA was analyzed by RT-PCR, and hCG binding level was determined by using the biotinylated hCG for a probe ligand.
Experiment 2 was undertaken to evaluate the effects of the phosphodiesterase inhibitor IBMX (Sigma) on meiotic resumption of oocytes and LHR formation in cumulus cells of COCs cultured with FSH. COCs were cultured with 0, 0.5, or 1 mM IBMX in 20 ng/ml FSH in maturation medium. After cultivation of COCs, nuclear status, the expression of LHR mRNA, and hCG binding level were evaluated.
Experiment 3 was conducted to identify the role of newly synthesized LHR in cumulus cells of COCs. COCs were precultured in medium supplemented with 20 ng/ml FSH and 0.5 mM IBMX for 20 h and then further cultured with 0, 1, or 10 µg/ml LH for 8 or 24 h. The level of cAMP in cumulus cells (8 h) and the production level of progesterone in cultured medium (8 and 24 h) were analyzed.
In experiment 4, after COCs were precultured in medium supplemented with 20 ng/ml FSH and 0.5 mM IBMX for 20 h and then further cultured with or without 1 µg/ml LH for 8, 16, or 24 h, nuclear status was examined. Some COCs cultured for 24 h with LH following 20 h of initial cultivation were used for IVF and subsequent IVC to the blastocyst stage. At 12, 48, and 144 h after IVF, proportions of pronuclear formation, cleavage rate, and blastocyst formation, respectively, were evaluated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We examined the time-dependent changes of the expression of LHR mRNA in cumulus cells of COCs cultured with FSH. Four types of amplified products (1121, 855, 411, and 185 bp) were detected in cumulus cells (Fig. 1). No significant differences in staining intensity for three types of products (855, 411, and 185 bp) were seen in cumulus cells from COCs cultured for up to 20 h (Fig. 1). The intesity of the largest amplified product (1121 bp) in cumulus cells of COCs remained low throughout the 8-h cultivation period; however, 12-h cultivation of COCs significantly increased the staining intensity for this product (Fig. 1). Subsequently, the intensity of this product further increased up to 20 h cultivation (Fig. 1).
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Time-dependent changes in the biotinylated hCG binding level of COCs cultured with 20 ng/ml FSH are shown in Figure 2A. The low hCG binding level was observed in COCs immediately recovered from the follicles or after 8 h of cultivation. Cultivation of COCs for 12 h led to a significant increase in the hCG binding level in the COCs. Thereafter, the level of hCG binding in COCs increased significantly up to 20 h of COC cultivation. Porcine LH effectively competed with biotinylated hCG for binding to COCs cultured for 20 h (Fig. 2B).
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Experiment 2
The majority of oocytes (85.0% ± 4.1%) before cultivation were arrested at the GV I or II stage (Fig. 3). The proportion of oocytes arrested at the GV I or II stage was significantly decreased in a time-dependent fashion (Fig. 3). After 20 h of cultivation, the proportion of oocytes arrested at these stages was only 15.0% ± 7.1%. However, the addition of IBMX into medium containing FSH suppressed the reduction in the proportion of oocytes arrested at the GV I or II stage (Fig. 3). Significant differences between the proportion of oocytes arrested at the GV I or II stage cultured in the presence and absence of IBMX were noted after 8 h of cultivation and up to 20 h (Fig. 3). After 20 h of cultivation with 0.5 or 1.0 mM IBMX, >80% of the oocytes were arrested at the GV I or II stage.
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The expression level of LHR mRNA in cumulus cells of COCs was not suppressed by the addition of 0.5 mM IBMX; however, expression was significantly suppressed by 1.0 mM IBMX (Fig. 4). The ligand binding assay also revealed that the binding level of hCG to COCs cultured with 0.5 mM IBMX and FSH for 20 h was comparable to that of COCs cultured with only FSH (Fig. 5). The cultivation of COCs with 1.0 mM IBMX for 20 h significantly decreased the level of hCG binding to COCs (Fig. 5).
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To arrest the oocytes at the GV stage without preventing the formation of LHR, the concentration of IBMX was adjusted to 0.5 mM in the maturation medium used for the other experiments.
Experiment 3
COCs were precultured for 20 h in medium supplemented with IBMX and FSH and then further cultured with 0, 1, or 10 µg/ml LH for 8 or 24 h. The level of cAMP of COCs cultured for 8 h with 1 or 10 µg/ml LH following 20 h of cultivation with FSH and IBMX was significantly increased as compared to that of COCs cultured for 8 h without LH following 20 h of cultivation (Fig. 6). The differences in cAMP level for cumulus cells cultured with 1 or 10 µg/ml LH were not significant.
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When COCs were cultured with 1 or 10 µg/ml LH for 8 h following cultivation with FSH and IBMX for 20 h, the level of progesterone in the medium was significantly higher than that in the medium where COCs were cultured without LH following 20 h of cultivation with FSH and IBMX (Fig. 7A). After 24 h of cultivation with LH, the level of progesterone was also significantly higher than that in the medium without LH (Fig. 7B). For the 8-h and 24-h cultivation periods, there was no significant difference in the level of progesterone in both LH treatment groups.
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Experiment 4
Meiotic progression of oocytes cultured in the presence of FSH and IBMX for 20 h and then with or without 1 µg/ml LH for an additional 8, 16, and 24 h is shown in Figure 8. After an additional 8 h of cultivation with LH, the majority of oocytes had reached the metaphase I (MI) stage at GVBD rates and the rate was significantly higher than those for oocytes cultured without LH. However, there was no significant difference in GVBD rates between the groups. At 16 h of cultivation with LH following the 20-h initial cultivation, the proportion of oocytes developed to the AT-I stage was 63.3% ± 6.2%. This rate was significantly higher than that of oocytes cultured without LH (26.7% ± 4.7%). There was no difference in maturation rate (85.0% ± 4.1% without LH, 90.0% ± 4.1% with LH) at 44 h of culture.
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At 12 h after insemination, there was no significant difference in the incidence of maturation, penetration, male pronucleus (MPN) formation, and monospermy between oocytes matured with and without 1 µg/ml LH following a 20-h initial cultivation in the presence of FSH and IBMX (Table 1). There was no significant difference in the rate of cleaved oocytes at 48 h after insemination (48.1–51.1%) (Fig. 9A). Blastocyst formation rate in oocytes matured in the presence of LH following 20 h of initial cultivation with FSH and IBMX was significantly higher (33.8% ± 5.5%) than that of oocytes cultured in the absence of LH (20.0% ± 3.5%) (Fig. 9A). The total numbers of blastocyst cells from oocytes cultured in the presence or absence of LH following 20 h of initial cultivation with FSH and IBMX during maturation differed significantly (52.7 ± 5.9 vs. 39.6 ± 6.2) (Fig. 9B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In porcine oocytes, a drop in the oocyte cAMP level was involved in the resumption of meiosis [8], and IBMX suppressed this decrease in oocyte cAMP [18]. In the present study, oocytes were synchronized to the GV II stage by exposure of COCs for 20 h to 0.5 mM IBMX, and the expression of LHR mRNA in cumulus cells was not suppressed. Jin et al. [26] reported that the conversion of cAMP to 5'AMP by phosphodiesterase type 4 was required for responsiveness to gonadotropin in mouse granulosa cells. In the present study, when COCs were cultured with 1.0 mM IBMX and FSH a low level of LHR mRNA expression in cumulus cells was observed. Therefore, because addition of a high concentration of phosphodiesterase inhibitor to the medium decreases hormone responsiveness in cumulus cells, the concentration of IBMX was adjusted to 0.5 mM in the maturation medium.
The newly synthesized LHR on cumulus cells functioned as a potent activator of cAMP production. The increase in cAMP in cumulus cells resulted in production of many potential factors that stimulate cumulus cell expansion and oocyte maturation [27–30]. Progesterone is secreted by folskolin-stimulated cumulus cells of porcine COCs through a cAMP-dependent pathway [31]. In the present study, when COCs were cultured with LH following cultivation with FSH and IBMX for 20 h, the level of progesterone in the medium was significantly increased concomitantly with the increase of cAMP level in cumulus cells. Osborn et al. [32] reported that the addition of aminoglutethimide into gonadotropin-containing medium resulted in low levels of progesterone and a rise in the proportion of ovine oocytes arrested at the GV stage. Progesterone mediates the close of gap junctional communication, which results from the reduction of connexin-43 in porcine cumulus cells [22] and in human myometrial cells [33]. The disruption of gap junctions within cumulus cells induces the meiotic resumption of rat and porcine oocytes because of the blockage of the conduction of meiosis-inhibitory signals from the outer layers of cumulus cells to the oocytes [34–37]. These findings suggest that progesterone secreted by COCs plays a role in inducing GVBD in porcine oocytes. Zhang and Armstrong [38] reported that when rat COCs were cultured in vitro with FSH and cytochrome P450scc inhibitor, progesterone secretion was almost completely inhibited and a low rate of fertilization of oocytes was observed. In pigs, the high concentration of progesterone secreted by COCs improves the rate of early embryonic development to the blastocyst stage after IVF [23]. In the present study, the high concentration of progesterone secreted by COCs cultured with LH accelerated meiotic progression to the MI stage and improved developmental competence to the blastocyst stage following IVF. Thus, the activation of newly synthesized LHR in cumulus cells increases cAMP levels and progesterone production in cumulus cells, resulting in an acceleration of oocyte meiotic progression and an increase of developmental competence to the blastocyst stage after IVF.
Problems encountered with IVP techniques for porcine embryos also may result from abnormal cytoplasmic maturation due to inappropriate IVM conditions. Nagashima et al [39] investigated the differences in developmental competence between porcine oocytes matured in vivo and in vitro. There was no significant difference in rates of fertilization; however, blastocyst formation rate was significantly lower for IVM oocytes. Moreover, the numbers of cells in blastocysts in vivo has been reported to be higher than those of IVP blastocysts [25, 40]. Grupen et al. [41] reported that the addition of epidermal growth factor (EGF) to maturation medium increased the number of cells in blastocysts, but the rate of blastocyst formation was very low (7%). Recently, a very useful IVM system was reported by Wu et al. [17]. They cultured porcine COCs with FSH, EGF, and butyrolactone 1 for 24 h and then with LH and EGF for 24 h. Treatment with butyrolactone 1 inhibited GVBD and allowed the oocytes to develop to the proper GV stage for LH stimulation, and after IVF, >40% of inseminated oocytes developed to the blastocyst stage. In the present study, treatment with FSH and 0.5 mM IBMX induced the expression of LHR in cumulus cells, held oocytes at the GV II stage, improved the rate of blastocyst formation, and increased the number of cells in blastocysts after IVF. Thus, the induction of LHR expression in cumulus cells while holding oocytes at the GV II stage is very important for producing IVM oocytes with high developmental competence. Further study is required to determine why LH induces cytoplasmic maturation of porcine oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Masayuki Shimada, Laboratory of Animal Reproduction, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan. FAX: 81 824 24 7988; mashimad{at}hiroshima-u.ac.jp
Received: 12 August 2002.
First decision: 4 September 2002.
Accepted: 8 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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