Introduction
In vitro embryo production through in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) has been possible in many kinds of mammalian species. Whale is an important mammalian animal. More information on reproductive events in follicular development, oocyte maturation, fertilization, and embryonic development should be obtained from male and female minke whale (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997a). Research on IVM, IVF and IVC of whale embryos will contribute to the basic understanding of reproductive physiology of cetaceans (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2008). Recently, whale oocytes have been used for cryopreservation, IVM, IVF and in vitro production of embryos.
Whale oocytes
Whale ovaries can be classified into three types according to the number and size of follicles. Type A has more than 200 follicles, and its diameter is <5 mm. The majority of the follicles are antral follicles. Types B and C have 50–200 and <50 follicles, respectively. The diameters of types B and C are up to 10 mm (Tetsuka et al., Reference Tetsuka, Asada, Mogoe, Fukui, Ishikawa and Ohsumi2004). Whale follicles can be classified into three types by their sizes. Small, medium and large follicles are <5, 5–10, and >10 mm in diameter, respectively. The diameters of oocyte ooplasm from the three follicle types are different, and there is no difference in the diameter of the whole oocyte and thickness of the zona pellucida among the three follicle types. The osmolarity of whale follicular fluid (wFF) from the three follicle types is 363.3–388.9 mOsmol. Follicular fluid from large follicles has a lower concentration of lactic acid than that from the small follicles, but the estradiol-17beta (E2) concentration of wFF increases as the follicle size increases (Nagai et al., Reference Nagai, Mogoe, Ishikawa, Hochi, Ohsumi and Fukui2007).
Whale oocytes are classified into four grades by the number of surrounding cumulus cells. Grades A and B have >5 and 1–3 layers of cumulus cells, grade C is naked oocyte or partially surrounded by cumulus cells, and grade D is surrounded by expanded cumulus cells (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997a). The recovery rates of cumulus–oocyte complexes (COCs) per sei and Bryde's whales are 16–30.6 and 6.7–26.8, respectively (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2008). The total proportions of grades A and B oocytes in the three follicle types are not different. The rates of germ vesicle (GV)-stage oocytes from the three follicle types are 55.9–72.1%. There are some degenerated oocytes in the small follicles. The rates of GV-stage oocytes are 25–81.1% for the four oocyte grades. Some grades C and D oocytes have resumed meiosis to metaphase II (MII) stage (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997a).
In vitro maturation of whale oocytes
Whale COCs can be matured in the medium in vitro, and the oocytes extruding the first polar body are considered to be mature. A portable CO2 incubator is a useful device for IVM of whale oocytes. The gas atmosphere of the portable incubator is 5% CO2 and 8–10% O2. The low oxygen tension reduces free oxygen radicals, and may promote cytoplasmic maturation and subsequent developmental competence of oocytes. The maturation rate of whale oocytes was 26.7% using the portable incubator (Iwayama et al., Reference Iwayama, Ishikawa, Ohsumi and Fukui2005).
Whale oocytes were cultured in a dish containing IVM medium supplemented with fetal whale serum (FWS), follicle stimulating hormone (FSH) and E2 for 96 h, and the maturation rate was 27.3% (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997a). Maturation rate of whale oocytes matured in medium with FSH/E2 for 30 h was 26.7% and the cumulus mass showed the maximum expansion (Iwayama et al., Reference Iwayama, Ishikawa, Ohsumi and Fukui2005). Adding FWS to the maturation medium improved the proportion of whale oocytes at MII stage, and whale COCs cultured in medium with 20% FWS had a maturation rate of 31.8% (Asada et al., Reference Asada, Tetsuka, Ishikawa, Ohsumi and Fukui2001a). Whale COCs can also be matured using the Well of the Well (WOW) method in medium supplemented with wFF, FSH, E2 and epidermal growth factor (EGF), and the maturation rate of oocytes cultured for 40 h was 30.4% (Fukui et al., Reference Fukui, Iwayama, Matsuoka, Nagai, Koma, Mogoe, Ishikawa, Fujise, Hirabayashi, Hochi, Kato and Ohsumi2007). Supplementation of wFF in IVM medium may improve maturation rate of whale oocytes due to containing beneficial hormones and growth factors in it. The IVM medium of whale oocytes can be adjusted to 390 mOsmol by addition of d-sorbitol. The increased osmolarity of medium may provide with physiological condition for oocyte maturation (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2008).
Cryopreservation of whale oocytes
Whale oocytes can be cryopreserved using ethylene glycol (EG).The morphologically viable proportion of post-thawed oocytes was 39.7%. Some damage is induced by the freezing and thawing procedures in the cryopreserved oocytes, such as rupture of the ooplasm membrane, vacuolation of microvilli, migration of cortical granules, and presence of vacuolated mitochondria. The presence of cumulus cells improves the proportion of oocytes at metaphase I, anaphase I, and telophase I stages. Thirty per cent of cryopreserved oocytes can resume meiosis in vitro, and four of 194 post-thawed oocytes can mature to the MII stage (Asada et al., Reference Asada, Horii, Mogoe, Fukui, Ishikawa and Ohsumi2000). Whale oocytes can also be vitrified using EG + dimethylsulfoxide, and the maturation rate after warming was 30.2%. Whale oocytes can be treated with cytochalasin B before vitrification, and the maturation rate after warming was 30.4% (Fujihira et al., Reference Fujihira, Kobayashi, Hochi, Hirabayashi, Ishikawa, Ohsumi and Fukui2006). Cryotop and open-pulled straw (OPS) have been used as the cryodevice for vitrification of whale COCs. The Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales (Iwayama et al., Reference Iwayama, Hochi, Kato, Hirabayashi, Kuwayama, Ishikawa, Ohsumi and Fukui2004).
In vitro fertilization of whale oocytes
The total length of whale spermatozoa is 56.7 μm. The common forms of whale sperm heads are conical and elliptic (Mogoe et al., Reference Mogoe, Fukui, Ishikawa and Ohsumi1998). Forty percent of spermatozoa frozen and thawed were motile, and 44% of spermatozoa frozen and thawed were vital. The motility and vitality of whale spermatozoa are correlated with the serum E2 levels (Fukui et al., Reference Fukui, Mogoe, Jung, Terawaki, Miyamoto, Ishikawa, Fujise and Ohsumi1996). Whale IVM oocytes can be fertilized in the fertilization medium with 20% FWS or 0.6% bovine serum albumin, sperm penetration and two-pronuclei formation occur in the whale oocytes (Asada et al., Reference Asada, Tetsuka, Ishikawa, Ohsumi and Fukui2001a). The rates of sperm penetration and pronuclear formation were higher in the whale oocytes matured for 120 h than in those matured for 96 h (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997b). Whale sperm pretreated with dithiothreitol (DTT) can be injected into IVM oocytes, and intracytoplasmic sperm injection (ICSI) embryos have the ability to develop to 2–4-cell stage (Asada et al., Reference Asada, Wei, Nagayama, Tetsuka, Ishikawa, Ohsumi and Fukui2001b).
In vitro development of whale embryos
Whale IVM oocytes can be injected with sperm, and two from 21 sperm-injected oocytes can develop to two-cell stage (Asada et al., Reference Asada, Wei, Nagayama, Tetsuka, Ishikawa, Ohsumi and Fukui2001b). No cleavage occurs in whale oocytes without insemination. Whale IVM oocytes inseminated with post-thawed spermatozoa were cultured with cumulus cells, and the inseminated oocytes cleaved to 2–16-cell stages (Fukui et al., Reference Fukui, Mogoe, Ishikawa and Ohsumi1997b). The cleavage, 4-cell and 8-cell rates of whale IVF embryos were not different between embryos from grades A and B oocytes. IVF embryos from whale grade B oocytes were cultured in FWS-supplemented medium, and 1.1% of the embryos developed to morula stage (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2008). Whale IVF embryos from grade A oocytes had higher cleavage and morula rates than those from grade B oocytes; 4.2% of grade A oocytes can develop to morula stage, but no morula can be formed from grade B oocytes (Asada et al., Reference Asada, Tetsuka, Ishikawa, Ohsumi and Fukui2001a).
Interspecies fertilization of whale sperm
Interspecies microinsemination has been used to examine the ability of whale haploid spermatogenic cells to induce Ca2+ oscillations. Whale round spermatids (RS), early-stage elongating spermatids (e-ES), late-stage elongating spermatids (1-ES) and testicular spermatozoa (TS) were injected into mouse oocytes, and the repetitive increases of intracellular Ca2+ concentration occurred in oocytes injected with the e-ES, 1-ES and TS. RS can not induce Ca2+ oscillations. Whale spermatogenic cells acquire sperm-borne oocyte-activating factor (SOAF) activity, which is related to their Ca2+ oscillation-inducing ability (Amemiya et al., Reference Amemiya, Hirabayashi, Ishikawa, Fukui and Hochi2007). SOAF activity in the whales is required during the early phase of spermiogenesis. Whale spermatid microinjected into mouse oocytes can induce the oocytes to be activated and resume meiosis. The RS can not activate mouse oocytes, but mouse oocytes can be activated by e-ES, l-ES and TS (Amemiya et al., Reference Amemiya, Iwanami, Kobayashi, Terao, Fukui, Ishikawa, Ohsumi, Hirabayashi and Hochi2004).
Whale spermatozoa centrosome introduced into bovine oocytes can contribute to the microtubule-organizing centre. Bovine mature oocytes injected with DTT-treated whale spermatozoa and activated with ethanol + 6-dimethylaminopurine formed a whale sperm aster. Assembly of the microtubule network is promoted by oocyte activation. The ratio of aster diameter to oocyte diameter is 0.57 in injected and activated oocytes (Kobayashi et al., Reference Kobayashi, Amemiya, Takeuchi, Tsujioka, Tominaga, Hirabayashi, Ishikawa, Fukui and Hochi2006).
Whale spermatozoa injected into mouse oocytes can lead to oocyte activation, and the sperm nucleus can transform into a male pronucleus (Watanabe et al., Reference Watanabe, Tateno, Kusakabe, Matsuoka, Kamiguchi, Fujise, Ishikawa, Ohsumi and Fukui2007). Whale frozen–thawed spermatozoa injected into bovine oocytes can participate in fertilization activities in bovine oocytes after the injection. Sperm head decondensation and male pronucleus formation occurred after ICSI. Male pronuclear formation rate was 39.1% in the injected oocytes. The development of male and female pronuclei is synchronous, and the mean diameters of male and female pronuclei are 30.4 μm and 29.3 μm, respectively (Wei & Fukui, Reference Wei and Fukui2000).
Whale interspecies somatic cell nuclear transfer
Whale interspecies somatic cell nuclear transfer (iSCNT) embryos can be reconstructed using bovine and porcine oocytes. Minke whale granulosa-cumulus cells (MWGC) and minke whale cumulus cells (MWCC) have been used as donor cells. Minke whale somatic cell nuclei transferred into enucleated bovine oocytes formed pseud-pronucleus (PPN), and the proportions of PPN were not different among interspecies and intraspecies SCNT oocytes. Minke whale iSCNT embryos had whale genomic DNA, and the embryos developed to 2–4–cell stages. There was no difference in the cleavage rates of minke whale iSCNT embryos from viable and dead cells (Ikumi et al., Reference Ikumi, Sawai, Takeuchi, Iwayama, Ishikawa, Ohsumi and Fuku2004). Sei whale iSCNT embryos have been reconstructed using whale fetal fibroblasts as donor nuclei and bovine oocytes as recipient cells. Both intracytoplasmic cell injection (ICI) and subzonal cell insertion (SUZI) are equally effective with respect to PPN formation and cleavage of sei whale iSCNT embryos. Bovine oocytes have the ability to support development of sei whale nuclei up to the 6-cell stage (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Lee, Hirayama, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2010). Sei whale iSCNT embryos can also be reconstructed using porcine oocytes. Porcine oocytes supported development of sei whale iSCNT embryos to the 4-cell stage, indicating that porcine oocytes can induce the nuclear reprogramming of sei whale somatic cells (Lee et al., Reference Lee, Bhuiyan, Watanabe, Matsuoka, Fujise, Ishikawa and Fukui2009). The high rates of developmental blockage in whale iSCNT might be due to failure in timely activation of the whale embryonic genome. Further studies are needed on reprogramming mechanism of whale nuclei at the molecular level to gain a better understanding of development in iSCNT embryos (Bhuiyan et al., Reference Bhuiyan, Suzuki, Watanabe, Lee, Hirayama, Matsuoka, Fujise, Ishikawa, Ohsumi and Fukui2010). Thus, development of iSCNT embryos may provide some information about whale embryo development.
