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RESEARCH |
Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan
Correspondence should be addressed to K Edashige; Email: keisuke{at}kochi-u.ac.jp
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Abstract |
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, 20β-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50–100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5–9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16–29%; hatching rate, 8–21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes. |
Introduction |
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In fish, including rainbow trout (Oncorhynchus mykiss; Jalabert 1976, Nagahama et al. 1980), goldfish (Carassius auratus; Jalabert 1976), yellow perch (Perca flavescens; Goetz & Theofan 1979), amago salmon (Oncorhynchus rhodurus; Nagahama et al. 1980), medaka (Oryzias latipes; Iwamatsu et al. 1987), and zebrafish (Danio rerio; Selman et al. 1993, 1994), 17, 20β-dihydroxy-4-pregnen-3-one (DHP) is effective as a maturation-inducing hormone for the in vitro maturation of fully grown oocytes at the GV stage (stage III). In addition, it has been reported that gonadotropin, activin, epidermal growth factor, and transforming growth factor- promote in vitro maturation of zebrafish oocytes (Pang & Ge 1999, 2002a, 2002b). It has also been reported that cyclin B (Kondo et al. 1997) and transforming growth factor-β1 (Kohli et al. 2003,2005) play roles in the maturation of zebrafish oocytes. In some fish, the pH of ovarian fluid is not neutral but alkaline (Fauvel et al. 1993, Lahnsteiner et al. 1995, Linhart et al. 1995). Moreover, Patiño et al. (2005) found that alkaline conditions facilitated the in vitro maturation of oocytes from the GV stage (stage III) to MII stage (stage V) in the Atlantic croaker (Micropogonias undulatus). However, none of these studies examined the ability of oocytes to be fertilized and to develop till hatching after in vitro maturation.
Only a few studies have examined the ability of matured oocytes to be fertilized and to develop till hatching. In the zebrafish, Li et al. (1993) reported an in vitro maturation method for zebrafish oocytes at stage III in which oocytes could be matured and fertilized, and the fertilized eggs could develop to term. But this in vitro maturation method has been reproduced neither by other researchers nor by themselves, and its use has not become widespread. At present, the viability of zebrafish oocytes at stage III is assessed by morphological observation and/or trypan blue (TB) staining (Isayeva et al. 2004, Plachinta et al. 2004). In the medaka, Iwamatsu (1973) reported that immature oocytes after luteinizing hormone (LH) surge could mature, be fertilized, hatch, and develop into adult fish with an in vitro schedule. Thus, immature medaka oocytes after LH surge can be made to undergo cytoplasmic maturation in vitro. However, hatched juveniles have not been obtained from in vitro matured medaka oocytes derived from oocytes before LH surge. Thus, it is not clear whether the current in vitro maturation method for fully grown fish oocytes at the GV stage before LH surge supports the cytoplasmic maturation of the oocytes.
In this study, we first examined whether the current in vitro maturation method for zebrafish oocytes supports the cytoplasmic maturation of the oocytes by examining their ability to be fertilized and to develop till hatching. Because the results showed that the method did not support cytoplasmic maturation sufficiently, we tried to develop a reliable in vitro maturation system for zebrafish oocytes by examining their ability to be fertilized and to develop till hatching.
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Results |
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Figure 3 shows the ability of stage III oocytes to mature, to be fertilized (cleave), and to develop till hatching after being cultured for various periods. The rates of cleavage (4%) and hatching (2%) were low when the culture period was 220 min, but increased as the culture period was extended; the highest rates of cleavage and hatching were obtained after 260 min of culture (29 and 20% respectively), and both rates decreased when the culture period was extended further (270–360 min). In this condition, the highest rates of cleavage and hatching remained low. Moreover, the period that supported the high rates of fertilization and term development was limited to 10 min.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Leibovitz L-15 medium was used as the basic medium for oocyte maturation because this amino acid-rich medium was considered reliable. To clarify the components essential for reliable in vitro maturation of oocytes, the use of simple balanced saline solutions (Kondo et al. 1997, Sakai et al. 1997, Kohli et al. 2003, 2005, Patiño et al. 2005) might be suitable. However, we used Leibovitz L-15 medium, because the aim of our study was to find conditions suitable for normal cytoplasmic maturation.
The most suitable concentration of Leibovitz L-15 medium was 90%, at which the osmolality was 0.29 Osm/kg (Fig. 2). This osmolality would be close to the isotonic value of zebrafish oocytes, although the isotonic osmolality of zebrafish oocytes has not been determined. The osmolality is close to that of the culture medium (0.27 Osm/kg, 90% TCM199) for medaka oocytes (Iwamatsu et al. 1987).
The pH of the maturation medium markedly influenced normal maturation of zebrafish oocytes, and the optimum pH was 9.0 (Fig. 4). Only a few studies have used alkaline culture media for the maturation of fish oocytes/follicles. Goetz & Nagahama (1985) reported in human chorionic gonadotropin (hCG)-treated goldfish that there was a significant increase in the spontaneous ovulation in vitro of fully grown follicles as the pH of culture media increased from 7.3 to 8.9. Patiño et al. (2005) reported in the Atlantic croaker that, in a culture medium containing hCG, the rates of spontaneous ovulation of fully grown follicles and GV breakdown of intrafollicular oocytes were significantly higher at pH 8.5 than 7.5. However, no attention has been paid to the pH of culture media for cytoplasmic maturation of fish oocytes. On the other hand, it has been reported that the pH of ovarian fluid is high in various species of fish, such as turbot (Scophtalmus maximus; pH 8.1; Fauvel et al. 1993), salmonid fish (pH 8.4–8.8; Lahnsteiner et al. 1995), Atlantic croaker (Micropogonias undulatus; pH 8.5; Patiño et al. 2005), and common carp (Cyprinus carpio L.; pH 9.0; Linhart et al. 1995). Therefore, alkaline conditions would be physiological for immature teleost oocytes and thus might be suitable for the in vitro maturation of oocytes.
When oocytes were matured in 90% Leibovitz L-15 medium at pH 9.0, the duration for which they retained the ability to be fertilized and to develop till hatching was extended. By incorporating BSA in the culture medium, the duration was not affected but the ability of oocytes to be fertilized and to develop till hatching was improved significantly. Here, most of the fertilized oocytes developed till hatching. Therefore, the addition of BSA to a maturation medium would be effective for acquiring the fertilizing ability in zebrafish oocytes. Sakai et al. (1997) reported that BSA was effective in maintaining the fertilizing ability of ovulated zebrafish oocytes at stage V for 1 h. It was also reported that ovarian fluid of coho salmon (Oncorhynchus kisutch) was effective in maintaining the fertilizing ability of ovulated zebrafish oocytes for 6 h (Corley-Smith et al. 1996). Thus, the ovarian fluid of fish might contain BSA-like proteins that affect the interaction between oocytes and sperm, and BSA might have similar effects on the interaction.
In this study, BSA was proven effective for the cytoplasmic maturation of zebrafish oocytes as for mammalian oocytes. Zebrafish oocytes that matured in 90% Leibovitz L-15 medium (pH 9.0) in the presence of BSA looked yellow, whereas those that matured in medium without BSA looked translucent (data not shown). Since yellowish oocytes at stage V are regarded as good quality oocytes (Westerfield 1995), BSA would be effective for cytoplasmic maturation (Fig. 5). In the medaka, it has also been reported that BSA is effective for the maturation of oocytes after LH surge (Iwamatsu 1973). In this study, we did not use endogenous proteins in fish ovarian fluid but used an exogenous protein, BSA. Therefore, the rates of cleavage and hatching could increase further if the essential component of fish ovarian fluid was used for the maturation of zebrafish oocytes. In bovine embryos, it has been suggested that BSA is endocytosed; then, after intracellular hydrolysis, its constituent amino acids contribute to nitrogen metabolism in the embryos (Orsi & Leese 2004). In zebrafish oocytes, the mechanism of the effect of BSA on maturation is not known, but it is possible that BSA is also endocytosed into fish oocytes. Further studies are necessary to clarify the mechanism by which BSA exerts a beneficial effect on the cytoplasmic maturation of zebrafish oocytes.
Unfortunately, only a small proportion (
10%) of oocytes were ovulated from the follicle in the present in vitro maturation system (data not shown). It has been reported that hCG plus activin (Pang & Ge 1999, 2002a) and epidermal growth factor plus transforming growth factor- (Pang & Ge 1999) are effective for the in vitro maturation of zebrafish oocytes. Using these substances for in vitro maturation with the proper timing, it may be possible to increase the ovulation rate.
Since juveniles having the wild-type appearance were obtained from wild-type sperm and in vitro matured leopard-type oocytes (Fig. 6B), sperm would have entered through the micropyle normally to fertilize in vitro matured oocytes. Therefore, the micropyle would have been constructed normally. If so, the present in vitro maturation system (alkaline solution containing BSA) may be close to physiological condition for the maturation of zebrafish oocytes.
Because the in vitro maturation method developed here was proven reliable, the method is effective for the practical use of immature zebrafish oocytes, and it may be applied to other teleost species as well. The present protocol may also become a powerful tool for understanding the mechanism regulating oogenesis and folliculogenesis in zebrafish. Since fish oocytes are large cells and have a large volume of cytoplasm, fish oocytes could be a suitable model for isolating factors controlling the cytoplasmic maturation of vertebrates' oocytes, especially at the molecular level.
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Materials and Methods |
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All experiments were approved by the Animal Ethics Committee of the College of Agriculture, Kochi University.
Assessment of the ability of zebrafish oocytes to mature, to be fertilized (cleave), and to develop till hatching
It has been reported that a 60 or 75% Leibovitz L-15 medium containing 1 µg/ml DHP (pH 7.5) is effective for the maturation of stage III zebrafish oocytes (Selman et al. 1993, 1994). Thus, we first cultured stage III oocytes in 4 ml of a 60 or 75% Leibovitz L-15 medium supplemented with 100 µg/ml gentamycin in a culture dish (35x10 mm). To make the two media, Leibovitz L-15 medium (100%) was diluted with distilled water. To induce maturation of the oocytes, 5 µl ethanol containing DHP (0.8 µg/µl) was added to 4 ml of the culture medium; the final concentration of DHP being 1 µg/ml. After incubation at 26 °C for 270 min, the maturation and viability of the oocytes were examined. In this study, we considered oocytes to have matured to stage V when their visible remnants of GV completely disappeared and they became maximally translucent, while they were not ovulated from the follicular membrane. To examine the proportion of viable oocytes, cultured oocytes were stained for 10 min in 60 or 75% Leibovitz L-15 medium containing 0.2% TB at 25 °C. Numbers of total oocytes and viable oocytes (unstained) were counted under a dissecting microscope.
To conduct IVF, the follicular membrane of matured oocytes at stage V was removed with a pair of fine forceps in the culture medium under a dissecting microscope (Fig. 7). The defolliculated oocytes were put on the lid of a culture dish containing 200 µl of the culture medium. A male zebrafish was killed by decapitation under anesthesia with tricaine solution. Two testes were recovered from the male and placed in 100 µl of Hank's medium (Westerfield 1995) in a culture dish covered with paraffin oil at 25 °C. Sperm were obtained by tearing the testes with forceps and a needle in the medium and the sperm suspension was cooled with ice (0 °C) to preserve activity. For the insemination of defolliculated oocytes, 10 µl of the sperm suspension was added to the culture medium (200 µl) containing defolliculated oocytes. After 1 min, inseminated oocytes were transferred to 4 ml E3 medium (Brand et al. 2002), and incubated at 26 °C. We also used oocytes matured in vivo as a control. The oocytes were observed under a microscope at 2 h after insemination for cleavage to the two-cell stage and at 72 h for hatching.
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To examine the effect of the culture period in 90% Leibovitz L-15 medium (pH 7.5) on the cytoplasmic maturation, stage III oocytes were cultured in 90% Leibovitz L-15 medium containing DHP (pH 7.5) for various periods ranging from 220 to 360 min at 26 °C.
To examine the effect of the pH of 90% Leibovitz L-15 medium on the cytoplasmic maturation, stage III oocytes were cultured in 90% Leibovitz L-15 medium containing DHP at various pH for 260 min at 26 °C. The pH was adjusted to 7.5, 8.5, 9.0, and 9.5 with NaOH.
To examine the effect of BSA and the culture period on the cytoplasmic maturation, stage III oocytes were cultured in 90% Leibovitz L-15 medium containing DHP (pH 9.0) in the presence or absence of 0.5% BSA for various periods ranging from 220 to 480 min at 26 °C.
The ability of cultured oocytes to be matured and fertilized and the ability of fertilizing eggs to develop till hatching were examined as described above.
Confirmation of the fertilization of zebrafish oocytes
Zebrafish oocytes could develop till hatching parthenogenetically (Streisinger et al. 1981, Westerfield 1995, Corley-Smith et al. 1996). To confirm the fertilization of in vitro matured oocytes, in vitro maturation and IVF were conducted using leopard-type (recessive) zebrafish oocytes. The stage III oocytes were collected from leopard-type zebrafish, and cultured in 90% Leibovitz L-15 medium containing DHP (pH 9.0) and 0.5% BSA for 270 min at 26 °C. The oocytes were inseminated with leopard-type (control) or wild-type (dominant) zebrafish sperm, as described previously. Hatched fry were raised in 10-l aquaria under 14 h light:10 h darkness for 60 days at 28 °C, and the pattern of the appearance of the juveniles was observed.
Statistical analysis
The significance of the difference in the rates of maturation, cleavage, and hatching after each treatment was analyzed with a 
2-test. Differences at P<0.05 were considered statistically significant.
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received 13 September 2007
First decision 19 October 2007
Revised manuscript received 25 November 2007
Accepted 4 December 2007
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References |
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