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. No. of normal Others ovary (cm) of oocytes oocytes 1 Left side 3.0 × 2.2 16 14 2 Right side 2.5 × 2.1 23 20 3 2 Left side 2.0 × 1.5 31 30 1 Right side 2.1 × 1.3 27 24 3 Mean 2.4 × 1.8 48.5/female 44/female 22/ovaries * L × W denotes length and width.
Table 2. Maturation rate of bear oocytes cultured for 24 and 48 h Maturation No. of oocytes No. of oocytes No. of oocytes time (h) used matured (%) not matured (%)
24 17 3 (17.6)b 14 (82.4) 48 32 19 (59.4)a 13 (40.6)
a, b Values with different superscripts are significantly different (P<0.05).
Table 3. Comparison of the sizes of in vitro matured oocytes from the bear and bovine Species No. of oocytes Diameter of oocyte (mean ± SE, µm) used With a ZP Cytoplasm without a ZP
Bear 10 161.8 ± 6.0a 135.3 ± 7.5a Cow 20 150.7 ± 4.9b 118.7 ± 7.5b
a, b Values with different superscripts within the same column are significantly different (P<0.05).
develop into 2 and 4-cell stage embryos (Fig. 1 D). (51.1 ± 4.9/female) were recovered from the ovaries of 48 black bears, and 777 (32.3%) of them were excellent in quality. The oocytes were matured in Discussion vitro in TCM-199 supplemented with 10% FBS (Gibco, 26140-079), 1 µg/ml estradiol-17β, 10 µg/ The ursidae family exhibits a number of unique ml FSH, 0.6 mM cysteine, and 0.2 mM Na-pyruvate, but little-understood reproductive characteristics. which is typically used for bovine oocyte For example, giant pandas (Ailuropoda melanoleuca) maturation. After 48 h of culture, 59.4% of the have adopted a strategy of delayed embryo oocytes were mature, although their cumulus cell implantation [15]. Despite the general lack of did not expand with the bovine oocyte control. The delayed implantation, there is little information morphology and appearance of the bear oocytes available that is focused upon bear oocytes. were similar to darkly pigmented dog oocytes [16]. In this study, we euthanized two female brown In a previous study [14], the highest incidence of bears and obtained their ovaries. The mean ovarian MII stage achievement during in vitro incubation size was 2.4 × 1.8 cm (range: 2.0–3.3 × 1.5–2.2 cm), was between 48 and 60 h. The reproductive cycles which is morphologically similar to dog ovaries of the donors (anestrous vs. luteal/pregnant) had [16]. When the ovaries were dissected, they yielded no influence on the oocyte maturation rate at 24 or 97 oocytes (an average of 48.5/female), and 88 48 h of cultures, but more of the oocytes recovered (90.7%) of them were morphologically classified as from anestrous females (43.9%) at 60 h reached the normal. Similar results were reported in a study MII stage than oocytes recovered from their luteal/ using black bears [14]. In the study, 2,403 oocytes pregnant counterparts (23.1%; P<0.05). The IN VITRO MATURATION OF BROWN BEAR OOCYTES 689 diameters of the matured bear oocytes with a ZP then establishment of pregnancy in a species other (161.8 ± 6.0 µm) and with cytoplasm without a ZP than the nuclear donor, may be the only way to (135.3 ± 7.5 µm) were significantly larger than those clone endangered animals. The giant panda is a of the bovine oocytes (150.7 ± 4.9 and 118.7 ± 7.5 highly endangered species and a member of the µm, respectively) and were similar to the size of Urasidae family. The giant panda has aroused tiger oocytes (P<0.05) [17]. worldwide interest [26]. However, it is impractical We attempted to activate the matured bear to clone this type of endangered species by oocytes at 48 h, and 31.6% of the activated oocytes interspecies cloning because there are insufficient developed to the 2 or 4-cell stage; however they numbers of oocytes and surrogate animals. It may were then blocked at the 2 or 4-cell stages. be possible to produce cloned giant panda embryos Although CR-1/2 media have been widely used for using interspecies nuclear transfer methods, brown in vitro production of bovine embryos [18], they do or black bear oocytes, and giant panda somatic not seem to be adequate for the culture of bear cells. Giant panda cubs may be produced by embryos. More extensive research of such things as transfer of cloned giant panda embryos into electro-activation conditions and in vitro culture surrogate mothers. media is needed in regard to bear oocytes. In summary, we have documented the feasibility The production of fertile oocytes by IVM would of bear oocyte IVM and electro-activation, but little not only allow rescue of oocytes destined for atresia information is currently available on IVF, embryo but also rescue of genetic material from valuable or culture, and oocyte activation. This technique must endangered species, such as dead or dying animals. be fully developed for “oocyte rescue” to become a This approach, in combination with IVF and viable approach in ursid conservation. embryo transfer, has resulted in the birth of young in laboratory rodents and several domestic species [19–22]. Recently, successful cloning of gaur [23, Acknowledgements 24] and mouflon [25] has demonstrated that the technique of interspecies somatic cell nuclear This study was supported by KOSEF (grant cloning can be practically applied to save highly #M10525010001-05N2501-00110). These endangered species. Interspecies cloning, which experiments were conducted under the approval of involves transfer of the cell nuclei of one species the Wildlife Institute of S. Korea. into the enucleated oocytes of another species and
References
1. International Union for the Conservation of Nature 5. Komatsu T, Yamamoto Y, Tsubota T, Atoji Y, (IUCN). available from
10. Platz CC, Jr, Wildt DE, Howard JG, Bush M. of tiger oocytes: a case report. Korean J Emb Trans Electroejaculation and semen analysis and freezing 2004; 19: 185–189. in the giant panda (Ailuropoda melanoleuca). J Reprod 18. Roenkrans CF, Zeng GQ, Mcnamara GT, Schoff Fertil 1983; 67: 9–12. PK, First NL. Development of bovine embryos in 11. Ishikawa A, Matsui M, Sakamoto H, Katagiri S, vitro as affected by energy substrates. Biol Reprod Takahashi Y. Cryopreservation of the semen 1993; 49: 459–462. collected by electroejaculation from the Hokkaido 19. Schroeder AC, Eppig D. The developmental brown bear (Ursus arctos yesoensis). J Vet Med Sci capacity of mouse oocytes that matured 2002; 64: 373–376. spontaneously in vitro is normal. Dev Biol 1984; 102: 12. Ishikawa A, Matsui M, Tsuruga H, Sakamoto H, 493–497. Takahashi Y, Kanagawa H. Electroejaculation and 20. Crister ES, Leibfried-Rudedge ML, Eyestone WH, semen characteristics of the captive Hokkaido Northey DL, First NL. Acquisition of brown bear (Ursus arctos yesoensis). J Vet Med Sci developmental competence during maturation in 1998; 60: 965–968. vitro. Theriogenology 1986; 25: 150. 13. Kojima E, Tsuruga H, Komatsu T, Murase T, 21. Moor RM, Trounson AO. Hormonal and follicular Tsubota T, Kita I. Characterization of semen factors affecting maturation of sheep oocytes in vitro collected from beagles and captive Japanese black and their subsequent developmental capacity. J bears (Ursus thibetanus japonicus). Theriogenology Reprod Fertil 1977; 49: 101–109. 2001; 55: 717–731. 22. Mattioli M, Bacci ML, Galeati G, Seren E. 14. Johnston LA, Donoghue AM, Igo W, Simmons LG, Developmental competence of pig oocytes matured Wildt DE. Oocyte recovery and maturation in the and fertilized in vitro. Theriogenology 1989; 31: 1201– American black bear (Ursus americanus): a model for 1207. endangered ursids. J Exp Zool 1994; 15: 269: 53–61. 23. Lanza RP, Cibelli J, Diaz F, Moraes C, Farin PW, 15. Hodges JK, Bevan DJ, Celma M, Hearn JP, Jones Farin CE, HammerCJ, West MD, Damiani P. DM, Kleiman DG, Knight JA, Moore HDM. Cloning of an endangered species using interspecies Aspects of the reproductive endocrinology of the nuclear transfer. Cloning 2000; 2: 79–90. female giant panda (Ailuropoda melanoleuca) with 24. Marshall A. Cloning for conservation. Nat Biotechnol special reference to the detection of ovulation and 2000; 18: 1129. pregnancy. J Zool 1984; 203: 253–267. 25. Loi P, Ptak G, Barboni B, Fulka J, Cappai P, 16. Sokolowski JH, Zimbelman RG, Goyings LS. Clinton M. Genetic rescue of an endangered Canine reproduction: reproductive organs and mammal by cross-species nuclear transfer using related structures of nonporous, parous and post-mortem somatic cells. Nat Biotechnol 2001; 19: postpartum bitch, Am J Vet Res 1963; 34: 1001–1013. 962–964. 17. Lee HS, Yin XJ, Lee YH, Min WK, Kim TS, Choi 26. Saegusa A. Mother bears could help save giant JW, Yoon BC, Kim JI, Kong IK. In vitro maturation panda. Nature 1998; 394: 409.