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In Vitro Maturation of Oocytes Derived from the Brown Bear (Ursus Arctos)

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In Vitro Maturation of Oocytes Derived from the Brown Bear (Ursus Arctos) Journal of Reproduction and Development, Vol. 53, No. 3, 2007 —Research Note— In Vitro Maturation of Oocytes Derived from the Brown Bear (Ursus Arctos) Xi-Jun YIN1), Hyo-Sang LEE1), Eu-Gene CHOI1), Xian-Feng YU1), Gye-Young PARK1), Inhyu BAE1), Chul-Ju YANG1), Dong-Hwan OH1), Nam-Hung KIM2) and Il-Keun KONG1) 1)Department of Animal Science and Technology, Sunchon National University, Suncheon, JeonNam 540-742 and 2)Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea Abstract. This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete “rescue” techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 × 1.8 cm (range: 2.0–3.3 × 1.5–2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 µg/ml estradiol-17β, and 10 µg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 µsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 ± 6.0 and 135.3 ± 7.5 µm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 ± 4.9 and 118.7 ± 7.5 µm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages. Key words: Activation, Brown bear (Ursus arctos), In vitro maturation, Oocyte size (J. Reprod. Dev. 53: 685–690, 2007) here are eight extant ursid species distributed detrimental genetic and demographic effects. worldwide. All eight species are endangered, The brown bear (Ursus arctos), a member of threatened, or vulnerable in all or part of their Ursidea family, is currently listed as threatened on ranges. It is well established that wild species are the International Union for the Conservation of highly susceptible to the combined pressures of Nature (IUCN) red list [1]. Oocyte collection and habitant loss and reduced population size, the maturation are important means of preserving result being potential extinction because of genetic resources. Endangered and threatened wild animals are now attracting attention as genetic Accepted for publication: January 7, 2007 Published online: February 19, 2007 resources and for species conservation, and semen Correspondence: I. K. Kong (e-mail: [email protected]) has been recovered from various captive animals. 686 YIN et al. Endocrinological and morphological studies have cumulus cells or a corona radiate). Only normal clarified the timing of puberty and breeding season oocytes were used for IVM. of female Japanese black bears [2–5]. There are several reports about techniques for collecting and IVM of oocytes cryopreserving the semen of ursids [6–13], but The basal maturation medium was TCM-199 these reports mainly focus on comparison of semen supplemented with 10% FBS (26140-079; Gibco, research; oocyte collection and in vitro maturation Grand Island, NY, USA), 1 µg/ml estradiol-17β, 10 (IVM) have only been reported in one paper [14]. µg/ml FSH, 0.6 mM cysteine and 0.2 mM Na- Investigation of oocyte characteristics and pyruvate. Bear cumulus-oocyte complexes (COCs) development of IVM methods are the next basic were rinsed three times in TL-Hepes. The oocytes steps for application of advanced reproductive were placed in four-well culture dishes containing technologies, such as in vitro fertilization (IVF), pre-equilibrated IVM medium and cultured in a 5% embryo transfer, and somatic cell cloning, in this CO2 incubator at 38.5 C for 24 or 48 h. As a control, species. It is difficult to obtain oocyte samples from bovine COCs were cultured in the same maturation bears due to limited access to the animals. No medium for 24 h. information is available for fundamental oocyte characteristics. Assessment of maturation and size of oocytes In order to determine whether meiotic After 24 or 48 h of IVM, cumulus cells were maturation can be induced in ovarian oocytes from moved into D-PBS supplemented with 0.1% the brown bear, we obtained ovaries from two polyvinyl alcohol (PVA-PBS) and 0.1% brown bears, collected oocytes, and investigated hyaluronidase by gentle pipetting using small-bore IVM timing. The sizes of matured oocytes were glass pipettes. Only oocytes with an evident first measured, and the developmental abilities of polar body and MII plate were considered mature. oocytes activated 48 h after IVM were evaluated. The sizes of these oocytes were determined using the diameters of those oocytes having a ZP or cytoplasm without a ZP and were compared with Materials and Methods matured bovine oocytes. Unless otherwise stated, all chemicals were Activation of bear oocytes and in vitro culture obtained from Sigma-Aldrich Chemical (St. Louis, The oocytes were equilibrated in 0.28 M mannitol MO, USA). solution containing 0.1 mM Ca++ and then transferred to an electrofusion chamber containing Ovary recovery and oocyte collection the same solution. Activation was induced by Ovaries were recovered from 2 female brown applying two 20 µsec DC pulses of 2.0 kV/cm using bears (Ursus arctos) euthanized on September 15, an Electro Cell Fusion Generator (Nepagene, 2006 at the Bugok Hawaii Amusement Animal Park Ichikawa, Japan). After electrical stimulation, the in S. Korea. Each bear was 15 years old and housed oocytes were cultured in TCM-199 containing 2 separately. The ovaries were excised immediately mM 6-dimethylaminopurine for 4 h. The activated and placed into phosphate buffered saline (D-PBS) oocytes were cultured in CR (Charles Rosenkrans) supplemented with 100 IU/ml penicillin G and 50 solution with 3 mg/mL fatty acid-free BSA (termed µg/ml streptomycin for transportation. The CR-1 medium) for 3 days and then CR solution plus ovaries were transported to the laboratory in PBS at 10% FBS (termed CR-2 medium) for the remaining 39 C and processed within 4 h. The size of the 4 days of culture. Embryo development was ovaries was measured before they were placed in assessed for cleavage and blastocyst formation rate petridishes containing TL-Hepes for further after 48 h and 7 days of in vitro culture, respectively. dissection. The ovarian surface tissue was sliced repeatedly with a razor. Oocytes were classified as Statistical analysis normal (medium to darkly pigmented and The sizes of oocytes were evaluated by Student’s completely or partially surrounded by either t-test. The ovaries and maturation rates to MII cumulus cells or a tight corona radiate) or other were analyzed by chi-square test. The level of (abnormal in shape, pale in color, or lacking significance was set at P<0.05. IN VITRO MATURATION OF BROWN BEAR OOCYTES 687 Fig. 1. Morphology of ovaries and oocytes in a brown bear. A) Ovaries of a brown bear. B) Oocytes cultured in vitro for 48 h. C) Matured oocytes with a first PB (arrow). D) The 2- and 4-cell stage embryos from activated oocytes. Results were 17.6 and 59.4% at 24 and 48 h IVM, respectively (Table 2). The diameters of the Bear ovary size and oocyte collection matured bear oocytes with a ZP (161.8 ± 6.0 µm) Bear ovaries (Fig. 1) were obtained from two 15- and with cytoplasm without a ZP (135.3 ± 7.5 µm) year-old female brown bears at the anestrous stage. were significantly (P<0.05) larger than those of the The lengths and widths of the ovaries were bovine oocytes (150.7 ± 4.9 and 118.7 ± 7.5 µm, measured. The mean ovarian size was 2.4 × 1.8 cm respectively; Table 3). (range: 2.0–3.3 × 1.5–2.2 cm). The ovaries obtained from the bears yielded 97 oocytes (an average of Oocyte activation in bears 48.5 per female), and 88 (90.7%) of them were Development to the cleavage stage of oocytes morphologically classified as normal. activated after 48 h of maturation culture was 31.6% (6/19); however all of the cleaved oocytes IVM of bear oocytes and oocyte size underwent complete lysis by 7 days after The oocytes were placed in IVM medium and cultivation. These results indicate that bear oocytes cultured in a 5% CO2 incubator at 38.5 C for 24 or 48 can develop to the MII stage (Fig. 1 C) in an in vitro h (Fig. 1). The maturation rates of the bear oocytes culture system and that activated oocytes can 688 YIN et al. Table 1. Ovary sizes and oocyte numbers for the bears Bears Source of Size (L × W)* Total no.
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