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Embryo |
Department of Obstetrics and Gynecology, Rikshospitalet University Hospital, Oslo 0027, Norway
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, early development, embryo, leptin, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leukemia inhibitory factor (LIF) is a STAT3-activating cytokine that may directly regulate early embryonic development [4]. Indeed, LIF secretion by the endometrium was shown to peak around implantation [5, 6], and mRNA transcripts of the LIF-receptor gene (LIFR) were detected in blastocyst-stage embryos [7–9]. In vitro, LIF was shown to promote blastocyst hatching, trophoblastic outgrowth, metalloproteinase secretion by trophoblasts, and implantation of cultured mouse embryos [7, 10, 11]. The effect of LIF on earlier stages of development, however, has been more controversial [11–13]. Nevertheless, LIF action on the embryo is not absolutely necessary for early development, because mouse embryos with a targeted disruption of the LIFR gene or gp130 gene (gp130 is a signaling protein that associates itself with the active LIFR) are able to develop beyond the implantation stage [14, 15]. In addition, it has been shown that even though maternal expression of LIF is mandatory for implantation to occur [16], LIF is only needed to induce a receptive state of the uterus [17].
Recently, leptin, a hormone that activates STAT3 signaling, was also related to early embryo development. The OB gene product leptin was first described as an adipocyte-derived hormone, which signals to the hypothalamus and regulates food intake and energy homeostasis (for review, see [18]). In its target cells, leptin activates specific leptin receptors (OB-R), and at least the OB-Rb long-receptor form triggers STAT3 activation [19]. The importance of leptin in reproduction is elucidated by several observations. First, the ob/ob mice that have a truncated form of leptin are sterile [20]. Exogenous leptin can restore fertility of ob/ob female mice [21], but pregnancy is disrupted by stopping leptin treatment during the first 4 days of embryo development [22]. Second, a direct effect of leptin on the embryo is suggested by the following: Leptin is present in the uterine fluid around implantation [23], leptin is secreted by endometrial cells [24], embryos express mRNA transcripts for OB-Rb after the morula stage, and leptin stimulates the development of mouse embryos in vitro, as was recently demonstrated by Kawamura et al. [23].
The simultaneous expression of OB-R and LIFR genes by blastocyst-stage embryos and the similarity in the signaling pathways of leptin and LIF prompted us to examine the concurrent effects of leptin and LIF on the rates of development, apoptosis, and activation of STAT3 signaling by mouse embryos in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C57BL/6JBom female (age, 7–9 wk) and B6CBAF1 male (age, 10 wk) mice were obtained from M&B A/S (Ry, Denmark). Animals were free from ecto- and endoparasites, mycoplasms, and pasteurellae; were supplied in filter boxes; and were acclimatized for 1 wk at the Department of Comparative Medicine, Rikshospitalet (Oslo, Norway). Mice were housed in Makrolon III (Scanbur BK, Nittedal, Norway) cages (male mice individually, female mice at six to eight per cage apart from the day of mating) on softwood granules as bedding. Room temperature (21 ± 1°C) and humidity (50% ± 1%) were regulated, and fluorescent lighting was on from 0700 to 1900 h, with 30-min on-off dimming. Mice had free access to standard pellet diet (B&K Universal AS, Nittedal, Norway) and tap water. Ethical approval was granted by the Approved Competent Person at Rikshospitalet as imposed by the Regulations of Animal Experimentation in Norway.
Superovulation and Embryo Recovery
Female mice were superovulated by i.p. injection of 5 IU of eCG in 0.9% NaCl (Folligon; Intervet, Boxmeer, The Netherlands) at 1300 h, followed by i.p. injection of 5 IU of hCG in 0.9% NaCl (Pregnyl; Organon, Oss, The Netherlands) 48 h later. They were then caged individually with a male mouse, and the presence of a vaginal plug the following morning confirmed successful mating (Day 1 of pregnancy).
On the morning of Day 2, plugged female mice were killed by cervical dislocation. Fallopian tubes were dissected and flushed with M2 medium (Sigma-Aldrich Norway AS, Oslo, Norway). Groups of 10–15 two-cell embryos with normal morphology (i.e., equal-sized blastomeres and no apparent fragmentation observed under an inverted light microscope) were transferred into 50-µl droplets of culture medium (see below) under paraffin oil (Sigma) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Embryo Culture and Observations
Culture media, leptin, and LIF Embryos were cultured in M16 medium (Sigma) without leptin and LIF (control) or in M16 medium with added mouse recombinant leptin (final concentrations, 1.6, 16, and 160 ng/ml; Sigma) and/or mouse recombinant LIF (final concentration, 200 ng/ml; biological activity, 50–500 U/ng, as measured by the manufacturer using the M1 mouse leukemic cell assay; Sigma). The 1.6 ng/ml concentration of leptin represents low serum leptin concentrations seen in 2- to 3-mo-old C57Bl/6J female mice fed with normal diet; the 16 ng/ml concentration corresponds to high serum leptin concentrations seen in the same mice fed with the high-fat diet [25].
The LIF concentration of 200 ng/ml was chosen for the following reasons: In earlier reports, doses up to 100 ng/ml [10] or 10 000 U/ml [12] of murine recombinant LIF were tested on the development of mouse embryos in vitro, but no maximal stimulation or plateau of effect was observed at these doses. In two initial experiments, we tested the effect of 2 and 20 ng/ml of LIF concentrations, but no difference was observed in the rate of blastocyst development, hatching, or cell number compared to the effect of 200 ng/ml of LIF or control (data not shown). Therefore, the 200 ng/ml dose was used subsequently.
Nineteen experiments were performed. In each experiment, 10–40 embryos (one to three culture droplets) were observed per treatment, always including several control cultures.
Observations Embryos were observed daily under an inverted microscope, and the number of embryos reaching the 4- to 8-cell stages, morula stage, and blastocyst stage by Days 3, 4, and 5, respectively, were recorded. In 14 experiments, blastocysts were removed from the cultures on Day 5 and were subjected to TUNEL assay or immunofluorescence (see below). In five experiments, blastocysts were cultured until Day 6, and the number of hatching/hatched blastocysts was established. The total number of cells in Day 6 blastocysts was determined by a modification of the method of Tarkowski [26]. Briefly, embryos were exposed to 0.3% sodium citrate for 2–3 min, fixed on a glass slide with 20% acetic acid:80% ethanol (v/v), and stained with orcein. Bright-field microscopic images were captured by a video camera in a Macintosh computer (Apple, Cupertino, CA), and nuclei were counted. The identity of the images was concealed before counting.
Visualization of DNA Fragmentation
The DNA fragmentation of cells was visualized by the TUNEL assay using the Fluorescein Apoptosis Detection System (Promega, Madison, WI). The TUNEL reaction detects strand breaks of DNA generated principally by apoptotic signals [27].
The procedure was as follows: Day 5 blastocysts were washed four times in 1% polyvinylpyrrolidone (PVP) in PBS, fixed with 4% paraformaldehyde for 1 h, and washed three times in 1% PVP in PBS. Fixed blastocysts were allowed to attach to poly-L-lysine-coated slides for 24–48 h. On the day of the assay, slides were washed twice in PBS, permeabilized in 0.2% Triton X-100 for 15 min, and washed twice in PBS. Slides were exposed to DNase buffer (40 mM Tris-HCl [pH 7.9], 10 mM NaCl, 6 mM MgCl2, and 10 mM CaCl2) for 5 min. At this point, 20 U/ml of DNase I (Sigma) in DNase buffer (100 µl) were added to the slides assigned as positive and negative controls for TUNEL reaction, and 100 µl of DNase buffer were added to the remaining slides. Slides were incubated for 30 min at 37°C and were subsequently washed four times in dH2O. Thereafter, slides were exposed to 100 µl of equilibration buffer (Promega) for 10 min. Reaction buffer (45 µl of equilibration buffer, 1 µl of TdT enzyme, and 5 µl of nucleotide mix, which contains fluorescein-12-dUTP; all reagents from Promega) was subsequently added. The TdT enzyme was replaced with 1 µl of dH2O for the slides assigned to negative control of TUNEL reaction. Slides were incubated for 60 min at 37°C. Reaction was stopped by incubating slides with 2x SSC buffer (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate; Promega), followed by two washing steps in PBS. Finally, slides were incubated with 50 µg/ml of RNase A (Sigma) in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl for 40 min at 37°C. Slides were then exposed to 1 µg/ml of propidium iodide for 15 min and then washed with dH2O. Vectashield (Vector Laboratories, Peterborough, U.K.) was applied over the embryos, and the slides were covered by a coverslip.
Confocal microscopy and cell counting Images were taken with a Leica TCS SP (Leica Microsystems, Heidelberg, Germany) confocal laser-scanning microscope using a 40x objective and eight-bit digital resolution for 1024 x 1024 pixels. The microscope was adjusted to detect fluorescein (green channel) and propidium iodide (red channel) with maximal sensitivity. Complete z-series of images were taken to ensure that each nucleus was sampled; 10–20 sections per embryo taken with a 1- to 2-µm step size were usually sufficient. At this point, the identity of the embryos was concealed by random numbering.
Series of two-color digital images were reconstructed in the Object-Image software [28], and apoptotic and normal nuclei were counted. As a rule, nuclei showing strong green fluorescence (DNA fragmentation as detected by TUNEL reaction) and chromatin condensation or fragmentation were classified as apoptotic [27]. Unfragmented nuclei without green fluorescence were classified as normal. Total number of cells was the sum of the number of apoptotic and normal cells. Care was taken to count each nucleus only once, because the same nucleus usually appeared in multiple adjacent sections.
Immunofluorescence
Antibodies Polyclonal anti-STAT3 antibodies that were derived from rabbits immunized with a synthetic peptide from the C-terminus of human STAT3 (Ab-1) were purchased from Lab Vision (Fremont, CA). Polyclonal antibodies specific for phospho-STAT3 (Tyr705) or phospho-STAT1 (Tyr701) were produced by immunizing rabbits with synthetic phosphotyrosine peptides around Tyr705 of mouse STAT3 or Tyr701 of human STAT1, respectively. According to the manufacturer (Cell Signaling Technology, Beverly, MA), these antibodies do not cross-react with nonphosphorylated STAT3 or STAT1, phosphorylated tyrosine of other STAT proteins, or serine-phosphorylated STAT3. Goat anti-rabbit immunoglobulin (Ig) G (H+L), labeled with the fluorochrome Alexa488, was used as a secondary antibody (Molecular Probes, Eugene, OR).
Procedure In three experiments, Day 5 blastocysts were removed from the culture medium and were rinsed in 1% BSA in Tris-buffered saline (BTBS). Subsequently, groups of blastocysts were pipetted from droplets to droplets of reagents (50 µl) using a flame-pulled Pasteur pipette. Zona-enclosed embryos were briefly exposed to acidic Tyrode medium until the zona pellucida disappeared. Embryos were then rinsed in BTBS, fixed in 4% paraformaldehyde in PBS for 1 h at 4°C, rinsed again in BTBS, and permeabilized with 0.1% Triton X-100 and 0.1% NP-40 in PBS for 1 h at room temperature. Embryos were then washed twice in BTBS for 1 h at 4°C and incubated with primary antibodies overnight at 4°C. Primary antibodies were diluted 1:200 (anti-STAT3) or 1:50 (antiphospho-STAT3) in 0.1% Tween-20 in BTBS. On the following morning, embryos were rinsed three times in BTBS and 0.1% Tween-20 and were exposed to secondary antibody (diluted to 1:750 in 10% BSA and 0.1% Tween-20 in TBS) for 2 h. After three washes in 0.1% Tween-20 in BTBS, some embryos were incubated with 1 mg/ml of concanavalin A, which was labeled with the fluorescent dye Alexa350 (Molecular Probes). After a wash in 0.1% Tween-20 in BTBS, embryos were finally exposed to propidium iodide (25 µg/ml) and RNase A (200 µg/ml) in BTBS to counterstain nuclei. Stained embryos were mounted in Vectashield and were observed in fluorescent microscopes on the following day. In all experiments, negative- and positive-control specimens were stained parallel with embryos.
Controls Negative control embryos were processed as described above, but the primary antibodies were either omitted from the protocol or were replaced with antiphospho-STAT1 (1:50 dilution). In initial experiments, phospho-STAT1-specific fluorescence was undetectable in embryos. Negative controls, where primary antibodies were replaced with normal rabbit serum (Institute of Immunology, Rikshospitalet) or with normal rabbit IgG (Upstate Biotechnology, Lake Placid, NY) where not suitable, as rabbit serum and rabbit IgG in 1:200 dilutions were apparently binding to mouse embryos and produced diffuse, strong fluorescent signal (results not shown).
The NIH/3T3 mouse embryo fibroblast cells stimulated with interleukin-6 (anti-STAT3 and antiphospho-STAT3) or interferon-
(antiphospho-STAT1) were used for positive control and to establish the optimal dilution of antibodies (results not shown).
Microscopy Specimens were observed under epifluorescent illumination using a high-magnification (63x) objective and filter set for rhodamine (red), fluorescein (green), and 4',6'-diamidino-2-phenylindole (blue). The identity of the slides was concealed, and STAT3- or phospho-STAT3-specific green fluorescence intensity was scored by eye using an arbitrary scale from 0 (absent) to 3 (intense) in each embryo, both for the cell membranes and for the nuclei. For illustrations, images were captured with a monochrome digital camera (F-view; Soft Imaging System, Münster, Germany) and were recolored and overlayed in Photoshop (version 6.0; Adobe Systems, San Jose, CA). Selected specimens were also observed in a confocal laser-scanning microscope set for detecting fluorescence of Alexa488 dye and propidium iodide.
Statistical Analysis
The proportions of embryos reaching a given stage of development were compared with the z-test. In these analyses, P < 0.0025 was considered to be significant to account for the multiple group comparisons. The number of cells and the proportion of cells with fragmented DNA were analyzed with ANOVA using SPSS (version 10; SPSS, Inc., Chicago, IL). In case of a significant effect (P < 0.05), the group differences were further analyzed with the Bonferroni test. Fluorescence intensities were compared by the Mann-Whitney test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The majority of embryos developed normally to Day 3 (>95%) and Day 4 (>90%) by reaching the 4- to 8-cell stages and the morula stage, respectively; no significant difference was seen in the development of control, leptin-treated, or LIF-treated embryos (data not shown). Significantly fewer blastocysts developed by Day 5 in the presence of 1.6 ng/ml of leptin, and fewer blastocysts were hatching on Day 5 in the presence of 16 ng/ml of leptin, compared to control cultures (Table 1). Blastocyst formation by Day 5 was enhanced by 1.6 or 16 ng/ml of leptin and 200 ng/ml of LIF compared to cultures supplemented with LIF only. Higher concentration of leptin (160 ng/ml) had no significant effect on the rate of blastocyst development (Table 1).
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The proportion of blastocyst cells that showed DNA fragmentation on Day 5 (Fig. 1, A–D) was significantly higher in leptin-exposed embryos than in control embryos or embryos that were cultured in the presence of LIF or LIF and leptin (Fig. 1F) (ANOVA: F3,118 = 5.51, P = 0.001; Bonferroni test, P < 0.05 for all the three comparisons). The total number of blastocyst cells was not significantly different among the groups (Fig. 1E) (ANOVA: F3,118 = 1.50, P = 0.22).
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Among the embryos that were cultured until Day 6, significantly fewer reached the hatching blastocyst stage in the presence of 16 and 160 ng/ml of leptin compared to controls (Table 2). The rate of blastocyst hatching was similar in cultures supplemented with LIF and leptin compared to cultures supplemented with LIF only (Table 2). The cell number of leptin- and/or LIF-exposed Day 6 blastocysts, counted using the method of Tarkowski [26], was statistically not different (Table 2) (ANOVA: F7,126 = 0.53, P = 0.82).
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STAT3- and Phospho-STAT3-Specific Immunofluorescence in Embryos
In Day 5 blastocysts, strong STAT3-specific immunofluorescence was observed in the trophectoderm (TE), whereas STAT3-specific immunofluorescence was at background levels in the region corresponding to the inner cell mass (ICM) (Fig. 2, A–C). This distribution of STAT3 was not the result of an incomplete penetration of antibodies, because phospho-STAT3-specific immunofluorescence was detected both in TE and ICM with apparently similar intensity (Fig. 2, D–F). The distribution of STAT3 and phospho-STAT3 in TE and ICM was similar in control, leptin-treated, and LIF-treated embryos and was invariably observed in total of 51 (STAT3) and 68 (phospho-STAT3) specimens. Phospho-STAT1-specific immunofluorescence (n = 27) (Fig. 2H) or fluorescence of embryos that were not exposed to primary antibodies (n = 4) (Fig. 2K) were undetectable.
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The STAT3-specific and phospho-STAT3-specific fluorescence was colocalized with propidium iodide, indicating nuclear localization of antigens (Figs. 2, A–F, and 3, B, C, G, and H). In the TE, where the larger distance between nuclei allowed detailed observation of cell membranes at high magnification, STAT3-specific and phospho-STAT3-specific fluorescence was also colocalized with concanavalin A-binding sites in the cell membrane (Fig. 3, C, D, H, and I), as was observed in a total of 23 (STAT3) and 35 (phospho-STAT3) embryos.
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The STAT3 and phospho-STAT3-specific immunofluorescence intensity of cell membranes and nuclei were scored for individual embryos and showed that, in the cell membranes, phospho-STAT3-specific fluorescence was most intense in leptin-treated embryos, was less intense in control embryos, and was weak or absent in LIF-treated or leptin- and LIF-treated embryos (Fig. 4 and Table 3). Nuclear phospho-STAT3-specific fluorescence and STAT3-specific fluorescence intensities were similar among the groups (Fig. 4 and Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In leptin-exposed embryos, a distinct distribution of tyrosine-phosphorylated STAT3 was observed, which was characterized by an intense phospho-STAT3-specific immunofluorescence in the cell membrane. In control and LIF-exposed embryos, however, cell membrane-associated immunofluorescence of phospho-STAT3 was weak or absent. The STAT3 signaling is a dynamic event, comprising phosphorylation of STAT3 by cytokine receptor-bound Jak kinases on the cell membrane, dimerization and translocation of active STAT3 into the nucleus, gene activation by phospho-STAT3, and its subsequent dephosphorylation [2, 29]. Although our findings provide only a "snapshot" of STAT3 signaling in embryos, we wish to propose the following explanation of the results. The findings indicate a significant spontaneous and/or paracrine or autocrine activation of STAT3 in blastocysts, as shown by the presence of tyrosine-phosphorylated STAT3 in the nuclei of control embryos. This basal STAT3 activation may be the result of several cytokines and their receptors that were shown to be expressed by blastocysts at the mRNA and/or the protein level, such as interleukin-6, interferon-
, leptin, and LIF [30]. Exposure of embryos to leptin was associated with a marked increase of phospho-STAT3-specific immunofluorescence in the cell membrane, presumably indicating the activation of leptin receptors and receptor-associated Jak kinases. Subsequent phosphorylation of STAT3 may have resulted in changes of gene expression and brought about impaired embryo development and induction of apoptosis by leptin. In embryos that were exposed to high LIF concentration with or without leptin, phospho-STAT3 fluorescence in the cell membrane was weak or absent, indicating that Jak kinases underwent down-regulation, consumption, or inactivation, which may thus disrupt leptin-induced STAT3 signaling and modify the effect of leptin on embryo development and apoptosis.
A communication between signaling of leptin and LIF on the subcellular level would require that LIFR and OB-R proteins are present simultaneously in the same embryonic cells. By using polymerase chain reaction-reverse transcriptase technology, the presence of LIFR [7, 8] and OB-R [23] transcripts in whole-mouse blastocysts were confirmed. By using in situ hybridization technique, transcripts of the LIFR gene were found in the ICM of blastocysts, whereas gp130 transcripts were also found in the TE cells [8]. The STAT3 proteins were immunolocalized in TE, but not in ICM, of mouse blastocysts [31], whereas phospho-STAT3 was immunolocalized in both TE and ICM (present study). Simultaneous presence of LIFR, OB-Rb, and STAT3 signaling proteins by the same embryonic cells, however, has not yet been shown.
A recent report by Kawamura et al. [23] indicates that leptin increases the proportion of fast-developing embryos and increases the cell number of mouse blastocysts in vitro. These findings differ from ours, but they may possibly be explained by several methodological differences, such as the strain of mice, embryo culture medium, scoring of the rate of embryonic development, and concentration of exogenous leptin. In particular, these authors studied embryos of the IVCS strain that were cultured in human tubal fluid medium; culture conditions and/or assessment of embryo development were such that approximately 11%–30% of control embryos exhibited normal development, compared to approximately 80%–90% in our experiments. In addition, Kawamura et al. observed a stimulatory effect of leptin, mostly at 100 or 1000 ng/ml (but not at 1 or 10 ng/ml) of leptin; these high concentrations are approximately 30- and 300-fold, respectively, over the mean leptin levels measured in the uterine fluid [23]. The effects of supraphysiological leptin concentrations, however, may not be applicable to physiological leptin levels because of down-regulation of leptin receptors [32].
The present study confirms the observations of Antczak and Van Blerkom [31] that TE cells are rich, whereas ICM cells are poor, in STAT3 protein, as detected by anti-STAT3 antibodies not selective for the state of phosphorylation. However, the approximately similar abundance of phospho-STAT3 in the nuclei of TE and ICM (Fig. 2) suggests, first, that STAT3 signaling is indeed active in ICM and, second, that a large part of STAT3 proteins in the nuclei of TE are unphosphorylated (whereas such accumulation of unphosphorylated STAT3 in the nuclei is not a feature of ICM). In fact, experiments on various cell lines and primary cells suggest that up to 50% of the cells' total STATs reside constitutively in the nucleus in unphosphorylated form [33] and regulate the expression of genes other than those controlled by cytokine-induced phosphorylated STATs [34]. Therefore, the different amount of phosphorylated and unphosphorylated STAT3 in TE and ICM raises the possibility that gene regulation by STAT3 differs in TE and ICM.
Embryonic apoptosis may be triggered by a variety of harmful conditions, such as suboptimal culture medium [35], chromosomal aberrations [36], heat shock [37], high concentrations of glucose [38], lack of transforming growth factor [39], and excess of several hormones/cytokines, such as insulin, insulin-like growth factor I [40], or tumor necrosis factor [38]. Nonetheless, apoptosis also occurs during normal development of blastocysts, because programmed cell death helps to remove unnecessary cells; the incidence of this "background" cell death depends on the number of cells in the embryo and diminishes with progressing cell divisions [36]. We observed an increased incidence of apoptosis in leptin-exposed embryos but no significant difference in the total number of cells of the same blastocysts, suggesting that apoptosis was induced by leptin. This leptin-induced apoptosis was inhibited by LIF, which coincides with the antiapoptotic effect of LIF in other systems, such as in embryonic stem cells [41] and primordial germ cells [42]. The physiological significance of leptin-induced apoptosis in blastocysts, given the statistically significant but small increase in the proportion of apoptotic cells, remains to be determined.
Although it is too early to connect the present findings regarding mouse embryos to human biology, a direct effect of leptin on preimplantation embryo development may have clinical implications. It is noteworthy that obese women, who usually have high serum leptin concentrations, have a lower chance to conceive after in vitro fertilization and embryo transfer [43] and are at increased risk of early pregnancy loss [44], effects which are possibly related to high maternal concentration of leptin [45].
In conclusion, high concentrations of leptin impair development and hatching of cultured mouse embryos and induce apoptosis in blastocysts. These effects are modified by LIF, presumably by interfering in STAT3 signaling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Péter Fedorcsák, Department of Obstetrics and Gynecology, Rikshospitalet University Hospital, Sognsvannsvn 20, Oslo 0027, Norway. FAX: 47 23072940; peter.fedorcsak{at}klinmed.uio.no
Received: 7 May 2003.
First decision: 26 May 2003.
Accepted: 5 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1997 56:1088-1096[Abstract]This article has been cited by other articles:
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