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Reproductive Technology |
Institut National de la Recherche Agronomique,3 Biologie du Développement et Biotechnologie, 78352 Jouy-en-Josas, France
Clinic of Sterility, Department of Obstetrics and Gynecology,4 University Hospital of Geneva, 1211 Geneva 14, Switzerland
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Department of Obstetrics and Gynecology,5 Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, fertilization, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies have identified paternally linked differences in the fertilization process between bulls of high and low fertility that manifest themselves during the first cell cycle. We have demonstrated that a beneficial paternal effect from spermatozoa recovered from bulls of high in vitro fertility is manifested during the first G1 phase after fertilization. This beneficial effect is then evidenced by an earlier onset and a longer duration of the first DNA replication in both male and female pronuclei, which more importantly, translates into higher rates of blastocyst formation 7 days later [5]. In bulls with low and high fertility capability the percentage of fertilization and overall timing of pronuclear formation is the same, however, we have found that the absence of a beneficial effect in low fertility spermatozoa was linked to a delay in both pronuclei of the onset of the first DNA synthesis, which was subsequently shorter [5]. The origin of this paternal control has not as yet been characterized.
In mice, sperm entry followed by decondensation in zona-free oocytes is associated with an increase of glucose metabolism (through glycolysis and pentose phosphate pathway [PPP]), whereas it remains at a basal level in parthenogenetically activated oocytes [6]. The presence of the male pronucleus within the oocyte cytoplasm is also correlated with an increase in glucose metabolism [7]. Glucose metabolism occurs through a number of pathways, however, the PPP has been demonstrated to be activated shortly after sperm penetration into the oocyte in sea urchins [8] and in mice [6]. Glucose metabolized through glycolysis provides energy by generating ATP. The PPP generates NADPH, which is used in reductive reactions, and ribose 5-phosphate, which is precursor of nucleotide synthesis for subsequent DNA replication [6]. Its implication in developmental processes has not, however, been evidenced. The activity of the PPP may vary according to cell types. In mice, about 0.6% and 2.5% of glucose is metabolized through the PPP in spermatozoa and oocytes, respectively [6].
Previous studies of heterospecific in vitro fertilization models have proposed that the sperm nucleus might bring a catalytic component or trigger its production to promote DNA synthesis in the female pronucleus [9, 10]. The expression of the paternal genome is not, however, involved in the onset of the S phase [11], whereas the male chromatin remodeling seems to be a prerequisite before DNA replication in both pronuclei [12]. Using the bovine model, we have therefore examined whether the differences in the paternal contributions of low- and high-fertility spermatozoa will provide insights into the interrelationship between the paternal and maternal pronuclei during the first cell cycle. We have hypothesized that a shift in glucose metabolism pathways, triggered by the fertilizing spermatozoon, could be at the origin of the long-term paternal effect on the morula-blastocyst transition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Batches of cumulus-oocyte complexes were harvested from the ovaries of adult cows, and obtained from a local slaughterhouse. The contents of antral follicles measuring between 2 and 8 mm were aspirated and recovered in PBS medium at 39°C. Only good-quality oocytes (compact cumulus with more than two cell layers) were kept for in vitro maturation (IVM) and in vitro fertilization (IVF). Frozen semen samples were supplied by the Union Nationale des Coopératives d'Elevage et d'Insémination Artificielle (Maisons-Alfort, France). We used batches of frozen ejaculated spermatozoa from different bulls. Although spermatozoa gave the same percentages of fertilization, they were known to induce different times of onset of the first S phase and different rates of blastocyst formation [5]. Characteristics of spermatozoa with high in vitro fertility (from bulls A and B) and low in vitro fertility (from bulls C and D) are shown in Table 1.
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Oocyte In Vitro Maturation, In Vitro Fertilization, In Vitro Culture, and Parthenogenetic Activation
IVM and IVF were performed according to the standard technique routinely used in the laboratory [5]. Thawed spermatozoa from each bull were selected by swim-up and prepared according to the methods described by Parrish et al. [13]. Cumulus cells were removed by vortexing, and groups of matured oocytes were inseminated at a concentration of 106 spermatozoa/ml in fertilization Tyrode albumin lactate pyruvate (TALP) medium [13] supplemented with 10 µg/ml of heparin at 39°C and 5% CO2 in air. Presumptive zygotes were either cocultured in B2 medium (CCD Laboratory, Paris, France) over a monolayer of Vero cells for 7 days [5] or subjected to different treatments (see below). For parthenogenetic activation (PA), cumulus cells of matured oocytes were removed by vortexing, and activation was induced by exposure to ethanol (7% in M199 medium) for 5 min at 39°C and washed extensively before transfer to TALP medium.
Characteristics of Chromatin After Incubation With 6-Dimethylaminopurine
To investigate the initial relationship between the male and female pronuclei in fertilized bovine oocytes we used the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP). Differences in the level of protein phosphorylation, when comparing fertilized and activated bovine oocytes, are related to male pronucleus formation [14]. As a result, 6-DMAP inhibits male pronuclear formation [15]. Although the S phase is not inhibited by incubation with 6-DMAP in the oocytes of mice, sheep, or bovines after parthenogenetic activation [16], the onset of the S phase in the female pronucleus when male pronucleus formation is inhibited has never been specified. 6-DMAP (5 mM; Sigma, St. Louis, MO) and 5-bromodeoxyuridine (BrdU, 1 mM, Sigma) were added to the TALP medium at 3 hours postinsemination (hpi) (mean time of sperm penetration), 12 hpi (before the mean time of onset of the S phase), or just after PA. Presumptive zygotes were fixed in 2.5% paraformaldehyde at 18 hpi (or 15 h postactivation; hpa). The immunocytochemical treatment (fluorescein isothiocyanate [FITC] labeling) was performed according to the method described by Adenot et al. [17] for either the detection of the Sphase or the detection of hyperacetylated histone H4. Detection of the pronuclear envelope, using an antibody anti-lamine A/C (supplied by J.E. Fléchon), was performed according to the method described by Shehu et al. [18]. In every experiment, chromatin was also stained with propidium iodide (Sigma). Zygotes were observed using a confocal microscope (LSM-310, Zeiss, Germany). Percentages of fertilization and activation were assessed by the number of oocytes with pronuclei relative to the number of inseminated or activated oocytes. Percentages of zygotes in S phase were determined by the number of zygotes with at least one FITC-labeled pronucleus relative to the number of fertilized or activated oocytes.
Metabolic Measurement of Spermatozoa and Fertilized Oocytes
The thawed spermatozoa of each bull were selected by swim-up and cultured in TALP medium supplemented with 10 µg/ml of heparin at 39°C for 3 h to achieve capacitation before metabolic measurement. Glucose metabolism was measured in sperm suspensions containing 104 cells/3-µl incubation droplet of TALP medium. Metabolism was also measured with or without 6-DMAP (5 mM) in groups of five metaphase II oocytes, five parthenogenetically activated oocytes, or five oocytes fertilized with spermatozoa from bulls of high or low in vitro fertility. All the oocytes were free of cumulus cells. At 3 hpi (mean time of sperm penetration), the fertilized oocytes were gently pipetted to remove excess spermatozoa prior to measurement. Parthenotes, however, were assessed just after PA. After metabolic measurement, oocytes and zygotes were fixed in paraformaldehyde (2.5%) and stained with propidium iodide (Sigma) to determine whether the resumption of meiosis or fertilization had occurred.
The metabolism of glucose was measured essentially as previously described [19, 20]. The final glucose concentration of the incubation droplets was 1 mM (unlabeled plus labeled glucose). Glucose metabolism via glycolysis was measured by collecting 3H2O released from 5-[3H]glucose (15.6 Ci/mM; Amersham Rahn, Zurich, Switzerland). Tracer quantities of 5-[3H]glucose (250 µCi/ml; about 20 µM) were added to TALP medium supplemented with 1 mM unlabeled glucose. Glucose metabolism via the PPP was assessed by measuring the 14CO2 produced from 1-[14C]glucose (57 mCi/mM; Amersham). 1-[14C]glucose was added to glucose-free TALP medium at a final concentration of 1 mM (57 µCi/ml). Because 14CO2 can be released from 1-[14C]glucose by metabolism through the PPP but also in the Krebs cycle after glycolytic metabolism to 3-[14C]pyruvate, experiments were performed with 6-[14C]glucose (56 mCi/mM; Amersham), which does not generate 14CO2 through the PPP, to evaluate the oxidation of labeled glucose by the Krebs cycle. An active PPP is clearly demonstrated when 14CO2 generated with 1-[14C]glucose is significantly higher than14CO2 generated with 6-[14C]glucose. In the absence of such a difference, it cannot be concluded that the PPP is active or inactive [21].
Measurements were performed in a closed incubation system, essentially as described by Rieger et al. [19]. Gametes were incubated with radiolabeled glucose in 3-µl droplets and placed on the inner side of the lids of Eppendorf tubes previously filled with 1.5 ml of 25 mM NaHCO3 (three tubes per bull spermatozoa per radio element per replicate, three tubes per oocyte group per radioelement per replicate) in order to trap 3H2O and 14CO2 released from 5-[3H]glucose and 1-[14C]glucose or 6-[14C]glucose, respectively. Both TALP medium and NaHCO3 were equilibrated with 5% CO2 in air at 39°C before measurements. After the lids were fitted on the Eppendorf tubes, incubation lasted 3 h at 39°C to allow detectable metabolic measurements. Sham preparations containing no gametes but 3-µl droplets of medium supplemented with radiolabeled glucose were included in triplicate in each experiment. At the end of incubation, the NaHCO3 fraction was collected quickly in scintillation vials containing 200 µl of 0.1 M NaOH, which promoted the conversion of dissolved CO2 and bicarbonate into carbonate. After an overnight incubation, 10 ml of scintillation cocktail (Lumagel; Lumac, Groningen, the Netherlands) was added to the vials and the radioactivity was counted with a liquid scintillation analyzer (Packard, Rungis, France). The mean count per minute (cpm) of the sham preparations was subtracted from the mean cpm obtained for each batch of gametes or zygotes. The difference obtained, which was representative of the metabolism of the gametes, was divided by the total cpm of labeled glucose and multiplied by the total quantity of glucose present in the 3-µl droplet. The 14CO2 and the 3H2O productions were considered detectable when the cpm values obtained after gamete or zygote incubations exceeded the cpm of the sham preparations by at least 50%.
6-Aminonicotinamide Treatment
To inhibit the activity of glucose-6-phosphate dehydrogenase (G6PDH), which is the first enzyme of the oxidative branch of the PPP, 6-aminonicotinamide (6-AN, 1 mM; Sigma) was added at 3 hpi. The inhibitor was then removed by sequential washings according to the experimental protocol (i.e., at 6 hpi after metabolic measurement or before the determination of the timing of pronuclear formation). Characterization of the onset of the S phase and of embryo in vitro development was performed as previously described [5].
Experimental Design, Statistical Analysis
Oocytes from ovaries obtained at the slaughterhouse were pooled. All experiments were carried out in three replicates (one replicate per day). In each replicate, batches of oocytes were fertilized separately with spermatozoa from bulls of high or low in vitro fertility. Except for metabolic measurement, each batch contained 18 to 22 oocytes. Results are expressed as the mean ± SD. Data were analyzed by ANOVA after arcsine transformation for the percentages [22]. Mean values were compared between conditions and between replicates using the Scheffé test and the Bartlett test for the homogeneity of the variances.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first set of experiments was performed in order to specify the influence of male pronucleus formation on the onset of the S phase in the female pronucleus. This was achieved by determining the onset of the S phase in the female pronucleus 1) when the formation of the male pronucleus was inhibited by 6-DMAP, and 2) in the absence of male pronucleus (parthenogenetic activation and 6-DMAP exposure). Percentages of IVF were not affected when the phosphokinase inhibitor 6-DMAP was added at 3 or 12 hpi (data not shown). Following IVF with spermatozoa of high or low fertility (from bulls A and B vs. bulls C and D, respectively), all zygotes exposed from 3 to 18 hpi with 6-DMAP did not form male pronuclei (i.e., spermatozoa remained partially decondensed and spherical), whereas female pronuclei developed fully (Fig. 1A–C). The immunodetection of lamine A/C demonstrated that envelopes surrounded both male and female chromatin (Fig. 1A and E). The presence of hyperacetylated histones H4 was evidenced in female pronuclei (Fig. 1B and F). Hyperacetylated histones H4 were not detected in spermatozoa before fertilization (data not shown), but the detection in decondensed sperm nuclei showed the initiation of the protamine/histone replacement (Fig. 1B and F). The onset of the S phase, however, did not occur in female pronuclei (Fig. 1C and G). When zygotes were exposed to 6-DMAP from 12 to 18 hpi, formation of male pronuclei was not inhibited and 94% ± 4% of zygotes entered the S phase in a normal timing. In parthenogenetically activated oocytes, the incubation with 6-DMAP (from the activation to 15 hpa) did not inhibit pronuclear formations, and in contrast to the fertilized oocytes, 95% ± 3% of them entered the S phase (Fig. 1D and H).
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Effect of Male Pronucleus Formation on Glucose Metabolism of In Vitro-Fertilized Oocytes
To investigate the possible role of glucose metabolism in mediating the effect of the male pronucleus on the S phase, we first examined whether a sperm-dependent activation of glucose metabolism occurred during sperm chromatin remodeling, and whether it was influenced by bull fertility. Levels of metabolized glucose were high through glycolysis and were lower through the PPP (Fig. 2A and B, respectively). The presence of few spermatozoa bound to the oocytes did not interfere with the metabolic measurement of zygotes because glycolysis and PPP of 10 to 20 bound spermatozoa were extremely low in comparison with the zygote. The incubation of oocytes with 6-[14C]glucose gave cpm values that were not different from the values of the sham preparations, indicating that no detectable levels of 14CO2 were generated with this radio element.
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Levels of metabolized glucose were significantly increased in fertilized oocytes (P < 0.01) compared with that of oocytes in metaphase II and parthenogenetic oocytes. Furthermore, glucose metabolized through the PPP was not detectable in the absence of fertilization. Levels of glucose metabolized by oocytes through glycolysis and PPP were higher (P < 0.01) following IVF with spermatozoa of high in vitro fertility (from bulls A and B) than with spermatozoa of low in vitro fertility (from bulls C and D).
To ascertain whether male pronucleus formation influenced glucose metabolism, measurements were performed after incubation with 6-DMAP. We had already shown above that incubation with 6-DMAP inhibited male pronucleus formation and S phase in the fully grown female pronucleus. In the presence of 6-DMAP, there was a significant decrease in glycolysis and PPP activities following IVF with spermatozoa of high in vitro fertility (P < 0.01), whereas there was no influence of 6-DMAP with spermatozoa of low in vitro fertility or parthenogenetic activation (P > 0.05) (Fig. 2A and B). However, levels of glucose metabolism remained higher for fertilized oocytes than for parthenotes.
Thus, sperm penetration promoted a moderate increase in glucose metabolism independent of the male pronucleus formation and it was not influenced by the in vitro fertility of the bulls. The male pronucleus formation led to a high glucose metabolism up-regulation following IVF with spermatozoa of high in vitro fertility (from bulls A and B), but not spermatozoa of low in vitro fertility (from bulls C and D).
Glucose Metabolism of Capacitated Spermatozoa from High-Fertility or Low-Fertility Bulls
To investigate whether the differences in metabolic activity during fertilization were intrinsic to the type of spermatozoa entering the oocyte we measured glycolysis and PPP activities on capacitated spermatozoa. For each ejaculate (Fig. 3A), the level of glucose metabolized through glycolysis was high (from 0.85 to 1.96 fmol/h per spermatozoon). Glycolysis of the capacitated spermatozoa did not differ significantly between bulls (P > 0.05), and the in vitro fertility (bulls A and B versus bulls C and D) was not related to the level of glycolysis (P > 0.05). Evaluation of the PPP also failed to correlate with in vitro fertility. For bulls B and D, the 14CO2 recovered was exclusively generated by the 1-[14C]glucose metabolism and was indicative of a functional PPP (Fig. 3B and C). The ratio of the production of 14CO2 from 1-[14C]glucose to the production of 3H2O from 5-[3H]glucose was low for both bulls (B and D). For bulls A and C there was no measurable difference of 14CO2 generated with 1-[14C]glucose and 6-[14C]glucose (Fig. 3B and C), and we cannot conclude that the PPP was active or inactive in these bulls. The glucose metabolism of the fertilized oocytes (Fig. 2A and B) was not related to the glucose metabolism of the capacitated spermatozoa before IVF (Fig. 3A and C).
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Effect of the Inhibition of the PPP During the Male Pronucleus Formation
To determine whether the level of the PPP during male pronucleus formation affected the onset of the S phase and subsequent embryo development, we inhibited the PPP from 3 to 6 hpi with 6-AN exposure. The level of glycolysis was not modified by this exposure (1.55 and 1.60 pmol per oocyte per hour for bulls A and B, 0.92 and 0.95 pmol per oocyte per hour for bulls C and D) and was similar to previous results (Fig. 2A). During the 6-AN exposure, we also verified that the level of PPP was not detectable, and that the timing of pronuclear formation was not changed (P > 0.05; Table 1) regardless of the treatment or the bull. Critically, after IVF with spermatozoa of high in vitro fertility and 6-AN exposure, the onset of the S phase occurred as late as after IVF with low-fertility spermatozoa. The subsequent development to the blastocyst stage was impaired and was significantly lower than in the absence of 6-AN (P < 0.05; Table 1). After IVF with spermatozoa of low in vitro fertility, the 6-AN exposure did not modify any parameter, even though the level of PPP was not detectable.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Decondensing spermatozoa, or early male pronuclei are unable to synthesize DNA even when such nuclei are in the presence of competent oocyte cytoplasm [9]. In our study the onset of the S phase was prevented in the female pronucleus when the formation of the male pronucleus was inhibited, suggesting that a fully grown male pronucleus is a prerequisite for the onset of the S phase. However, the presence of hyperacetylated histones H4 and the assembling of the pronuclear envelope proved that the female pronucleus was able to initiate DNA synthesis [17, 23, 24]. Our results are consistent with those of Ramalho-Santos [12] who have reported that in rhesus monkey oocytes treated by intracytoplasmic sperm injection, male chromatin remodeling is required before replication of either parental genome, and indicate a unique G1/S transition checkpoint during zygotic interphase. Thus the sperm penetration triggers a particular pathway for the onset of the S phase, indicating an establishment of a functional relationship between both pronuclei. This pathway is different in parthenotes that have only one haploid pronucleus undergoing S phase independently of sperm factors [23].
When glucose metabolism was measured following IVF we found that both glycolysis and PPP activities were dramatically increased in fertilized oocytes and that the level of glucose metabolism was correlated with the ability of the spermatozoon to determine an early (high in vitro fertility) or a delayed onset of S phase (low in vitro fertility). The increase in glycolysis we observed in our study remained moderate (about 1.5 pmol/oocyte per hour) compared with the high glycolysis rate reported in bovine blastocysts (about 10 pmol/embryo per hour) [19]. In contrast to our results, Khurana and Niemann [25] did not detect differences of glycolysis from the in vitro-matured oocyte to the 12-cell stage in cattle. In the same species, metabolic measurements between 21 and 24 hpi did not show differences of glucose metabolism according to the developmental potential of the zygote [26]. This discrepancy may be explained by a transient increase in glucose metabolism during male pronucleus formation, which was not examined in previous studies. The negligible glucose metabolism observed in parthenotes compared to pronuclear formation, indicates that metabolic activation during pronuclear formation is induced by specific sperm factors that may promote the translation of mRNAs coding for glucose metabolic enzymes [27] or the availability of metabolic substrates such as glucose or glucose 6-phosphate [28]. The sperm penetration into the oocyte or its early decondensation is involved in the low glucose metabolic activation. In contrast, the up-regulation of the metabolic activation induced by high-fertility spermatozoa required a fully grown male pronucleus formation. The up-regulation of the PPP appeared relevant to the precocity of the S phase because its onset was delayed but not prevented in the presence of 6-AN during male pronucleus formation. Although a role of glycolysis cannot be entirely excluded, a high glycolysis activation, which was not abolished by 6-AN treatment, was not sufficient to induce early S phase in the absence of the activated PPP.
The metabolic products of the PPP involved in the onset of the S phase remain to be identified. In general, the PPP generates ribose 5-phosphate, which is a precursor of nucleotide synthesis for subsequent DNA replication, and NADPH, which is used in reductive reactions. Although the oocyte contains sufficient amounts of ribose 5-phosphate to support the S phase in parthenotes, a rapid accumulation of ribose 5-phosphate may be required for an early onset of the S phase. The inability of low-fertility spermatozoa to generate a high activation of the PPP may explain the delayed S phase and subsequent low embryo development. In addition, an increase in NADPH may activate NADPH-dependent enzymes such as NADPH oxidase, as demonstrated in sea urchins [29]. The developmental competence of the zygote can also be improved by the activation of glutathione reductase to increase reduced glutathione level [30]. As demonstrated in other cell systems [31], we may hypothesize that the redox potential (NADPH/NADP) may control the onset of the S phase: alternate pathways such as the malic enzyme or the NADP-dependent isocitrate dehydrogenase may produce NADPH but not as efficiently as the PPP, resulting in a delayed onset of the S phase.
There exists an interesting, paradoxical effect of glucose during bovine IVF. Although exogenous glucose is necessary for fertilization in mice [32] and is present in the oviduct and the uterus [33], it is not required for in vitro capacitation and the first cell cycle in bovines [34]. However, as already observed by Thompson et al. [35], bovine embryos use little endogenous substrates when appropriate exogenous substrates are present in the culture medium. In the absence of exogenous glucose, the source could be originated from glycogen contained in spermatozoa [36] or in the oocytes [37].
It is becoming more apparent that a key interrelationship exists between the male and female pronuclei to ensure that fertilization and embryo development occur in the correct manner. In this study we have shown that in bovine, male pronucleus formation was necessary to initiate the onset of the S phase in the female pronucleus. We propose that following IVF with spermatozoa of high in vitro fertility, male pronucleus formation is linked with the release or activation of elements via the PPP that determine the early onset of the S phase during the G1 phase. A critical, simple shift in a metabolic pathway of the zygote can lead to a long-lasting effect on blastocyst formation. The origins of these differences in sperm populations are unknown. Further experiments are needed to clarify whether artificially increasing PPP activity after IVF with low-fertility spermatozoa could improve the timing of the S phase and subsequent embryo development. It would be of interest to examine whether differences in the level of PPP after fertilization are also to be found in the human species, in which dramatic differences exist in the vitro fertility of spermatozoa from different donors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.P. Renard, INRA, Biologie du Développement et Biotechnologie, 78352 Jouy-en-Josas, France. FAX: 33 1 34 65 26 77; renard{at}jouy.inra.fr
Received: 23 September 2002.
First decision: 21 October 2002.
Accepted: 25 November 2002.
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REFERENCES |
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