Reprod Med Biol (2009) 8:19 24 DOI 10.1007/s12522-008-0003-8 ORIGINAL ARTICLE The beneficial effect of fructose and glucose on in vitro maturation and the fertilization of porcine oocytes H. Tsujii Æ J. H. Lee Æ M. S. Hossain Æ K. M. A. Tareq Æ K. Hamano Æ T. Sawada Received: 2 May 2008 / Accepted: 10 October 2008 / Published online: 13 December 2008 Ó Japan Society for Reproductive Medicine 2008 Abstract Purpose This study was conducted to investigate the effects of supplementing fructose in the culture medium on in vitro maturation (IVM), in vitro fertilization (IVF), and metabolism of porcine oocytes. Methods Porcine oocytes were matured in vitro in modified North Carolina State University 37 medium (NCSU- 37) and then supplemented with either glucose (5.5 mm), fructose (5.5 mm), or glucose (2.75 mm) plus fructose (2.75 mm). The maturation and fertilization of oocytes, and the incorporation and oxidation of 14 C-glucose, 14 C- fructose, and 14 C-methionine in oocytes at different stages of development were examined. Results The supplementation of glucose plus fructose significantly promoted (P \ 0.05) oocytes germinal vesicle break down (GVBD), maturation to metaphase II (MII), penetration by spermatozoa, and male pronuclear formation compared with glucose. The incorporation and oxidation of 14 C-methionine into the oocyte significantly increased (P \ 0.05) with glucose plus fructose supplementation than glucose. A significantly higher (P \ 0.05) rate of incorporation and oxidation was achieved with 14 C- fructose compared to 14 C-glucose. Conclusions Glucose plus fructose supplementation improved maturation, penetration by spermatozoa, male H. Tsujii (&) J. H. Lee M. S. Hossain K. M. A. Tareq K. Hamano Laboratory of Animal Biotechnology, Shinshu University, Minamiminowa-mura, Nagano 399-4598, Japan e-mail: htsujii@gipmc.shinshu-u.ac.jp T. Sawada Nagoya Reproduction Center, The Sawada Women s Clinic, Chikusaku, Nagoya, Aichi, Japan pronuclear formation, and energy metabolism by porcine oocytes. Keywords Fructose and glucose In vitro fertilization In vitro maturation Metabolism Porcine ooctye Introduction The environments in which oocytes are cultured during in vitro maturation (IVM) and in vitro fertilization (IVF) play an important role in the in vitro development of embryos. Several research groups have developed a range of culture media for porcine embryos, such as modified Whitten s medium [1], North Carolina State University 23 and 37 media (NCSU-23 and 37) [2, 3], Iowa State University (ISU) medium [4], and Beltsville embryo culture medium 3 (BECM-3) [5]. Among these media, NCSU-37 is now widely used for various purposes, such as for IVM or in vitro culture (IVC) of porcine embryos produced by either in vivo or in vitro fertilization. In any culture medium, the energy substrate is one of the most important ingredients for the optimum in vitro development of embryos. Glucose was included as an energy substrate in NCSU- 37 medium for IVM of porcine oocytes and subsequent IVC of in vitro produced embryos [6, 7]. Although pyruvate and lactate in the absence of glucose are able to support the in vitro development of bovine embryos [8], glucose is widely supplemented as a major energy substrate. In contrast with the metabolism of pyruvate and lactate, glucose metabolism in IVF embryos differs from their in vivo counterparts [9]. Glucose is also known to have a detrimental effect on early embryo development. Like glucose, fructose is capable of entering the glycolytic pathway. In addition, fructose is present in rabbits [10] and
20 Reprod Med Biol (2009) 8:19 24 bovine [11] reproductive tracts and effectively supports embryonic development in hamster [12], bovine [13, 14], mouse [15], porcine [16], and human embryos [17]. Furthermore, fructose can increase the total cell numbers in both hamster [12] and bovine blastocysts [13]. Moreover, the expression of the fructose-transporter gene in bovine embryos emphasizes the embryotrophic role of fructose [18]. The previous report showed that the supplementation of glucose plus fructose in mtalp medium improved the sperm functions potentialities of boar spermatozoa [19]. The role of fructose in early embryonic development is still unclear, and the metabolic activity influenced by fructose has not yet been reported elsewhere. Thus, the objectives of the present study were to determine whether fructose can be an alternative to glucose or the synergistic effect of glucose and fructose for supporting meiotic maturation in porcine oocytes, their subsequent development, and their metabolic activity. Materials and methods Oocyte collection and in vitro maturation Ovaries were obtained from crossbred prepubertal gilts of Landrace, Large White, and Duroc breeds at a local slaughterhouse and transported to the laboratory in physiological saline 0.9% (w/v) NaCl containing 75 lg/ml potassium penicillin G and 50 lg/ml streptomycin sulfsate maintained at 37 C. Cumulus-oocyte complexes (COCs) were aspirated from follicles (3 6 mm in diameter) using an 18-guage needle attached to a syringe. After being washed twice with phosphate-buffered saline, only oocytes with uniform ooplasm and compact cumulus cells were used. The selected COCs were transferred into a maturation medium-modified North Carolina State University 37 medium (mncsu-37) supplemented with 0.6 mm cysteine (Nacalai Tesque, Kyoto, Japan), 1 mm dibutyryl cyclic AMP (dbcamp; Wako, Osaka, Japan), 10 IU/mL equine chorionic gonadotropin (ecg; Sankyo Zoki, Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hcg; Sankyo Zoki), 50 lg/ml gentamicin (Nacalai Tesque) and 10% (v/ v) porcine follicular fluid (pff). The mncsu-37 medium was basically the same as the original formulation, except for the glucose or fructose (where each of 5.5 mm glucose, 5.5 mm fructose, and 2.75 mm glucose plus 2.75 mm fructose were used as treatments). About 30 COCs were cultured with 100 ll maturation medium under a layer of mineral oil in a four-well multi-dish (non-treated dish, Iwaki, Japan) for 22 h. They were then transferred to maturation medium without hormones and dbcamp cultured for an additional 22 h. All cultures were performed at 38.5 C in a humidified incubator containing 5% CO 2 in air. Sperm preparation and in vitro fertilization Freshly ejaculated semen from a single Duroc boar was obtained from a local experiment station (Nagano Animal Industry Experiment Station, Shiojiri, Nagano, Japan). The semen was washed twice with modified Tyrode s albumin lactate pyruvate (mtalp) medium [20] with 5.0 mm glucose and 0.5 mm fructose and then centrifuged at 7339g for 3 min. After centrifugation, the supernatant was removed and the sperm pellet was preincubated for 15 min at 38.5 C before fertilization. IVF was carried out in accordance with the method described by Kikuchi et al. [5]. After IVM culture for 44 h, COCs were washed three times with mtalp fertilization medium and transferred in a group of 30 COCs in 100 ll drops of IVF medium that had been covered with warm mineral oil in a four-well multi-dish. The dishes were kept in the incubator for approximately 30 min before spermatozoa were added. Preincubated spermatozoa were introduced into the fertilization medium drop containing matured COCs to give a final concentration of 1 9 10 6 / ml. Co-incubation was performed for 6 h at 38.5 C ina humidified incubator containing 5% CO 2 in air. After 6 h of co-incubation, oocytes were freed from the cumulus cells and attached spermatozoa, and were transferred into IVC medium. The basic IVC medium was NCSU-37 supplemented with 5.5 mm glucose, 5.5 mm fructose, or 2.75 mm glucose plus 2.75 mm fructose containing 4 mg/ ml BSA, 0.17 mm sodium pyruvate, 2.73 mm sodium lactate, and 50 lg/ml gentamicin that had been covered with warm mineral oil in a four-well multi-dish. Fresh culture was added to the putative embryo for 18 h at 38.5 C in a humidified incubator containing 5% CO 2 in air. Assessment of oocyte nuclear status and fertilization At the end of IVM culture, oocytes were denuded mechanically from cumulus cells in modified phosphatebuffered saline supplemented with 1 mg/ml hyaluronidase (Nacalai Tesque). Denuded oocytes were then mounted on a glass slide and fixed with acetic acid:ethanol (1:3, v/v) for 48 h. The fixed oocytes were stained with acetic-orcein (1% orcein in 45% acetic acid) and examined using a phase-contrast microscope (IX-50, Olympus, Tokyo, Japan) at 4009 magnification. Oocytes were observed to determine whether they underwent germinal vesicle break down (GVBD) or advanced to the metaphase II (MII) stage. Stained oocytes with an abnormal chromatin configuration or without chromatin were considered degenerated. On the other hand, at 18 h after IVF, presumptive zygotes were mounted on a glass slide, fixed, and stained, as described above. Oocytes containing both female and
Reprod Med Biol (2009) 8:19 24 21 male pronuclei were considered as fertilized and were categorized as normal or polyspermic, according to the number of swollen sperm heads and pronuclei in the cytoplasm. Incorporation and oxidation of radiolabel glucose, fructose, and methionine by porcine oocytes All radioactive substrates, 14 C-glucose (spec. act: 185 MBq/ mmol), 14 C-fructose (spec. act: 212 MBq/mmol), and 14 C-methionine (spec. act: 39 MBq/mmol), were obtained from Moravek Biochemicals, Inc., USA. Each of the five oocytes at a particular stage of development was transferred in a 100 ll drop of maturation medium containing 18.5 kbq 14 C-glucose, 14 C-fructose, or 14 C-methionine, then overlaid with mineral oil. 14 C-glucose and 14 C-fructose were incorporated into a glucose and fructose free medium with oocytes at first polar body (22 h), second polar body (44 h), and IVF (68 h) stages; 14 C-methionine was incorporated into medium supplemented with 5.5 mm glucose, fructose, or glucose plus fructose only at the MII stage of development. On other hand, 1 ml of 2.5 mm NaOH solution was transferred into a 1.5 ml micro tube as a trap for the evolved 14 CO 2. Both micro tubes of NaOH and 14 C-glucose as well as 14 C-fructose or 14 C-methionine with oocytes were confined into a scintillation vial using a rubber stopper. The scintillation vials were incubated for 0 to 3 h in a cell culture incubator with a humidified atmosphere of 5% CO 2 in air at 37 C. Just after completion of the specific incubation period, the metabolic reactions of oocytes were stopped with an injection of 100 ll of 10% perchloric acid (PCA) for glucose and fructose and trichloroacetic acid (TCA) for methionine kept at room temperature for 24 h. The acid insoluble materials were carefully washed by millipore filtration (8.0 lm white SCWP, 47 mm; Millipore Corporation, Bedford, MA, USA) with 5% PCA or TCA and the filter papers were kept overnight under a lamp. After drying, the filter papers were transferred into scintillation vials. The NaOH solution was transferred into a new scintillation vial by washing 3 4 times with cocktail (0.5% PPO? 0.03% POPOP solution in toluene). All the scintillation vials plus three blanks for each group with 5 ml of cocktail were set in a liquid scintillation counter (LS-6500, Beckman Instruments Inc., USA) to determine the levels of radioactivity. This experiment was conducted three times to improve its accuracy. The value of incorporation and oxidation was expressed directly as counts per minutes (cpm) [21]. Statistical analysis Each experiment was repeated four times. All percentage data were subjected to arc-sine transformation before statistical analysis. Statistical analyses were carried out using two-way analysis of variance (ANOVA). A post hoc procedure using Fisher s protected least significant difference test (PLSD test) was adopted for multiple comparisons between the groups where appropriate. For the statistical analysis, STATVIEW software (Abacus Concepts Inc., Berkeley, CA, USA) was used. Data were expressed as mean ± S.E.M. Differences of P \ 0.05 were considered significant. Results The effect of fructose and glucose plus fructose on the maturation of oocytes is shown in Table 1. The supplementation of glucose plus fructose significantly (P \ 0.05) increased the rate of GVBD and MII, as compared with glucose or fructose. The influence on in vitro fertilization of fructose and the combination of fructose and glucose is shown in Table 2. Compared with glucose, the supplementation of fructose and glucose plus fructose increased the percentage of fertilization of oocytes, where the effect of glucose plus fructose was statistically significant (P \ 0.05) than the others treatment. Furthermore, in the supplementation of glucose plus fructose, both the highest percentage of monospermic fertilization and the lowest percentage of polyspermic fertilization were observed. The effect of fructose and glucose plus fructose on the rate of male pronucleus formation according to fertilization is shown in Table 3. Compared to glucose, the supplementation of glucose plus fructose significantly increased (P \ 0.05) the rate of male pronucleus formation. The rate of incorporation and oxidation of 14 C-methionine in MII oocytes is shown in Table 4. The incorporation of 14 C-methionine was more efficient (P \ 0.05) in fructose and glucose plus fructose treated MII stage oocytes. The oxidation of 14 C-methionine in oocytes significantly increased (P \ 0.05) with only glucose plus fructose; the performance of fructose was higher than that with glucose. The rate of incorporation and oxidation of 14 C-glucose and 14 C-fructose in the development process of porcine oocytes are shown in Table 5. At the second polar body and IVF stages of development, the incorporation of 14 C- fructose was significantly higher (P \ 0.05) than that of 14 C-glucose. Whereas, the oxidation of 14 C-fructose significantly increased (P \ 0.05) compared to 14 C-glucose only in embryos after the IVF stage. Discussion In the present study, supplementing NCSU-37 medium with fructose or glucose plus fructose instead of glucose
22 Reprod Med Biol (2009) 8:19 24 Table 1 Effect of fructose and the combination of glucose and fructose on nuclear status in porcine oocytes Treatment No. of oocytes examined No. of oocytes at a specific stage GVBD (%) MII (%) Glucose (5.5 mm) 307 267 (87.0 ± 0.5) A 239 (78.0 ± 1.2) a Fructose (5.5 mm) 339 293 (86.5 ± 2.1) A 274 (81.3 ± 1.4) a Glucose (2.75 mm)? fructose (2.75 mm) 352 327 (92.3 ± 0.9) B 306 (87.0 ± 0.9) b Four replicate trials were conducted. Percentages are expressed as mean ± SEM GVBD germinal vesicle break down, MII metaphase II Values in parentheses indicate percentages. Values with different superscripts in the same column are significantly different (P \ 0.05) Table 2 Effect of fructose and the combination of glucose and fructose on penetration, monospermy, and polyspermy of porcine oocytes Treatment Oocytes fertilized Penetrated (%) Polyspermic (%) Monospermic (%) Glucose (5.5 mm) 132 (56.2 ± 2.1) a 59 (45.0 ± 1.5) 73 (55.0 ± 1.5) Fructose (5.5 mm) 164 (60.3 ± 2.3) a 72 (44.3 ± 3.2) 92 (55.7 ± 3.2) Glucose (2.75 mm)? fructose (2.75 mm) 214 (70.0 ± 1.5) b 83 (39.0 ± 2.5) 131 (61.0 ± 2.5) Four replicate trials were conducted. Percentages are expressed as mean ± SEM Values in parentheses indicate percentages. Values with different superscripts in the same column are significantly different (P \ 0.05) Table 3 Effect of fructose and the combination of glucose and fructose on in vitro male pronucleus formation of porcine oocytes Treatment No. of oocytes examined Male pronucleus formation (%) Glucose (5.5 mm) 306 117 (38.3 ± 2.5) a Fructose (5.5 mm) 337 138 (41.0 ± 1.0) a Glucose (2.75 mm)? fructose (2.75 mm) 348 193 (55.5 ± 2.0) b Four replicate trials were conducted. Percentages are expressed as mean ± SEM Values in parentheses indicate percentages. Values with different superscripts in the same column are significantly different (P \ 0.05) Table 4 Incorporation and oxidation of 14 C-methionine in porcine oocytes in vitro maturation (cpm/oocyte) Treatment Metaphase II Incorporation Oxidation Glucose (5.5 mm) 765.8 ± 12.5 A 22.7 ± 1.3 a Fructose (5.5 mm) 1,076.8 ± 25.2 B 26.0 ± 3.4 ab Glucose (2.75 mm)? fructose (2.75 mm) 1,350.5 ± 27.0 C 30.7 ± 1.3 b Four replicate trials were conducted. Values are expressed as mean ± SEM Values with different superscripts in the same column are significantly different (P \ 0.05) increased the rate of maturation in porcine oocytes. Glucose is an essential energy source for oocyte maturation and embryonic development, involving normal mechanisms regulating the nuclear and cytoplasmic maturation of oocytes and maintenance of the developmental competence of embryos [22 24]. Wongsrikeao et al. [16] observed that the supplementation of 5.5 mm glucose or fructose in NCSU-37 medium increased the ability of oocytes to reach MII. They observed that in the absence of hexose, the percentage of GVBD, MII, and monospermy were 81.5, 53.8 and 42.7%, respectively. Furthermore, there was no significant difference in the interaction between the hexose-type and the concentration. In this study, a similar trend of development with glucose or fructose was observed. Wongsrikeao et al. [16] reported that GVBD, MII, and monospermy improved to 93.8, 79.4, and 52.9%, respectively, with glucose (5.5 mm), and 92.0, 70.2 and 49.7%, respectively, with fructose (5.5 mm). It is worth mentioning that in the present study, the combination of glucose and fructose synergistically improved GVBD, MII, and monospermy to 92.3, 87.0, and 61.0%, respectively. Thus, the present combination approach specifically enhanced the percentages of MII and monospermy much more than that reported by Wongsrikeao et al. [16]. Wangsrikeao et al. [16] reported that oocyte maturation and fertilization and blastocyst formation did not significantly differ between fructose and glucose treatments and decreased the DNA fragment nucleus index of the
Reprod Med Biol (2009) 8:19 24 23 Table 5 Incorporation and oxidation of 14 C-glucose and 14 C-fructose in porcine oocytes in vitro maturation and fertilization incorporation of 14 C-glucose and 14 C-fructose (cpm/oocyte) Treatment Oocyte I a Oocyte II b After IVF Incubation time 22 h 44 h 68 h Incorporation of 14 C-glucose and 14 C-fructose 14 C-glucose 767.1 ± 19.8 841.3 ± 16.2 A 1,407.1 ± 25.5 a 14 C-fructose 880.4 ± 20.1 1,242.3 ± 23.8 B 1,867.0 ± 27.2 b Oxidation of 14 C-glucose and 14 C-fructose 14 C-glucose 14.7 ± 2.8 13.6 ± 0.9 20.6 ± 2.7 a 14 C-fructose 11.3 ± 0.6 17.0 ± 1.9 29.3 ± 1.0 b Four replicate trials were conducted. Values are expressed as mean ± SEM Values with different superscripts in the same column are significantly different (P \ 0.05) a Oocytes with first polar body b Oocytes with second polar bodies embryos. On the other hand, it was reported previously that supplementing mtalp medium with glucose plus fructose instead of glucose, improved the acrosome reaction and in vitro fertilization capability of boar spermatozoa [19]. This result might be due to fructose strongly activating glucose breakdown in glycolysis. This can be explained by the fact that porcine oocytes may utilize fructose without altering the activity of rate-limiting enzymes for glycolysis. During glycolysis, glucose is converted into fructose-1,6-biphosphate (F1,6BP) followed by degradation into lactate with the production of energy and NADH. However, fructose can enter the glycolytic pathway directly after conversion into F1,6BP which involves a lower energy requirement. Thus, the result of this study suggests that fructose metabolism through glycolysis is a primary factor for the progression of porcine oocyte nuclear maturation. The present results clearly demonstrated that the addition of fructose to the culture medium enhanced the male pronuclear formation of oocytes, particularly when they were cultured in the presence of glucose plus fructose. The results also suggest that the combination of glucose and fructose in the medium has the potential to increase monospermy and simultaneously decrease polyspermy. It was reported that glucose during oocyte maturation increased the contents of reactive oxygen species in oocytes and decreased glutathione levels, impairing the developmental competence of oocytes after fertilization [25]. Some researchers have suggested that this toxic effect is also related to the disruption of mitochondrial organization [26] and reduction of respiration [27], resulting in the arrest of embryonic development [28]. On the other hand, the present results are consistent with another study in which fructose showed advantages over glucose, including lower toxicity at high dose [12]and higher rates of cleavage and morula formation in bovine oocytes [29]. Wongsrikeao et al. [16] reported that fructose was a practical alternative to glucose for supporting IVM of porcine oocytes, and fructose was superior to glucose for producing high-quality porcine embryos in vitro. In some strains of mice (OF1 and C57Bl), fructose alone was not the best energy source [15], but in the Swiss strain, fructose eliminated the 2-cell block and resulted in higher blastocyst formation rates than glucose [30]. Thus, the present hypothesis of the synergistic effect of glucose and fructose in the culture medium might be a logical approach. Most recently, Bhuiyan et al. [31] reported that combined glucose and fructose supplementation in the culture medium significantly increased blastocyst formation in bovine transgenic cloned embryos. The expression of Glut5, which has a high affinity for fructose, indicates that the early embryo is capable of transporting this energy substrate [18]. The transcription onset by early embryo goes along with a high demand for nucleotides and ribose phosphate. The early cleavage stages of different species metabolize energy substrates mainly by the pentose-phosphate pathway and not by the Embden Meyerhof-pathway [32]. From the present results, it was supposed that a possible fructose uptake via Glut5 could coincide with shift from the pentose-phosphate pathway towards the production of ribose-5-phosphate, an essential precursor for nucleotide synthesis. In the present study, the incorporation efficiency of 14 C- methionine at MII stage was higher in glucose plus fructose treated oocytes. The degradation of amino acids convert them to either citric acid cycle intermediates or their precursors so that they can be metabolized into either CO 2 and H 2 O or used in gluconeogenesis. It was speculated that amino acids were the key regulators of oocyte metabolism and viability, and that the maturation of oocytes in a medium lacking these regulators results in an inability of the oocyte to control its metabolism. 14 C-fructose was metabolized more vigorously than 14 C-glucose at different developmental stages. This indicates that the incorporation and oxidation of glucose and fructose are predominant factors for oocyte metabolism and maturational potential. This also suggests that porcine oocytes have a tendency to meet their energy requirement from the exogenous hexose sources. Though there are previous reports on the utilization of radioactive methionine and glucose in Mongolian gerbil [33] and rat [34] embryos, respectively, there is little information on fructose utilization in porcine embryos. Nonetheless, Guixe et al. [35] proposed that fructose plays an important role in the utilization of 14 C-glucose in frog oocytes. Thus, in the present study, the increased incorporation of 14 C-fructose by porcine oocytes is an indicator of the increased metabolism of fructose via the glycolysis pathway. These data suggest that for developing embryos, ATP production from fructose is more convenient than from glucose.
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