Development of fresh and vitrified agouti ovarian tissue after xenografting to ovariectomised severe combined immunodeficiency (SCID) mice
Érica C. G. Praxedes A , Gabriela L. Lima B , Luana G. P. Bezerra A , Fernanda A. Santos C , Marcelo B. Bezerra C , Denise D. Guerreiro D , Ana P. R. Rodrigues D , Sheyla F. S. Domingues E and Alexandre R. Silva A FA Laboratory of Animal Germplasm Conservation, Universidade Federal Rural do Semi-Árido, BR 110, Km 47, Costa e Silva, 59625-900, Mossoró, RN, Brazil.
B Department of Animal Sciences, Instituto Federal de Educação, Ciência e Tecnologia do Ceará (IFCE), CE-292, KM 15, Gisélia Pinheiro, 63115-500, Crato-CE, Brazil.
C Laboratory of Gonadal Transplantation and In vitro Embryo Production, Universidade Federal Rural do Semi-Árido, BR 110, Km 47, Costa e Silva, 59625-900, Mossoró, RN, Brazil.
D Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculdade de Veterinária, Universidade do Ceará, Av. Paranjana 1700, Campus do Itaperi, 60714-903, Fortaleza, CE, Brazil.
E Laboratory of Amazon Wild Animals Biology and Medicine, Universidade Federal do Pará, Jaderlândia, 68746-360, Castanhal, PA, Brazil.
F Corresponding author. Email: legio2000@yahoo.com
Reproduction, Fertility and Development 30(3) 459-468 https://doi.org/10.1071/RD17051
Submitted: 9 February 2017 Accepted: 12 July 2017 Published: 8 August 2017
Abstract
The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (± s.e.m.) 20.6 ± 8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.
Additional keywords: hystricognath, preantral follicles, rodent, wildlife, xenotransplantation.
References
Abir, R., Fisch, B., Jessel, S., Felz, C., Ben-Haroush, A., and Orvieto, R. (2011). Improving post transplantation survival of human ovarian tissue by treating the host and graft. Fertil. Steril. 95, 1205–1210.| Improving post transplantation survival of human ovarian tissue by treating the host and graft.Crossref | GoogleScholarGoogle Scholar |
Aerts, J. M. J., Clercq, J. P. B., Andries, S., Leroy, J. L. M. R., Van Aelst, S., and Bols, P. E. J. (2008). Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after cryologic solid surface vitrification or slow-rate freezing. Cryobiology 57, 163–169.
| Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after cryologic solid surface vitrification or slow-rate freezing.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtFyltrrE&md5=3bc65d7aecbfce9ab089e4f7e6f317a2CAS |
Amorim, C. A., Jacobs, S., Devireddy, R. V., Van Langendonckt, A., Vanacker, J., Jaeger, J., Luyckx, V., Donnez, J., and Dolmans, M. M. (2013). Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue. Hum. Reprod. 28, 2146–2156.
| Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtFOgtr3L&md5=76b7045a395e9f72eade28f716fa0839CAS |
Bandeira, F. T., Carvalho, A. A., Castro, S. V., Lima, L. F., Viana, D. A., Evangelista, J., Pereira, M., Campello, C. C., Figueiredo, J. R., and Rodrigues, A. P. R. (2015). Two methods of vitrification followed by in vitro culture of the ovine ovary: evaluation of the follicular development and ovarian extracellular matrix. Reprod. Domest. Anim. 50, 177–185.
| Two methods of vitrification followed by in vitro culture of the ovine ovary: evaluation of the follicular development and ovarian extracellular matrix.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXktVemtLw%3D&md5=98955e0316adf5abdc43908ed73e2ec2CAS |
Barros, F. F. P. C., Teixeira, P. P. M., Silva, M. A. M., Silva, A. S. L., Medeiros, R. M., Coelho, C. M. M., Bandarra, M. B., Macedo, M. F., Bezerra, M. B., and Vicente, W. R. R. (2014). Xenotransplante em camundongos imunossuprimidos coletado por meio de ovariectomia unilateral total vídeolaparoscópica em ovelha. Rev. Bras. Reprod. Anim., Belo Horizonte 38, 121–126.
Bartoskova, A., Ondrackova, P., Leva, L., Vitasek, R., Novotny, R., Janosovska, M., and Faldyna, M. (2014). The effects of in vitro exposure to progesterone and estradiol-17β on the activity of canine neutrophils. Vet. Med. (Praha) 59, 202–209.
Bedaiwy, M. A., and Hussein, M. R. (2004). Histological evaluation and in situ localization of apoptosis in fresh and cryopreserved ovarian tissue. Middle East Fertil. Soc. J. 9, 1110–5690.
| Histological evaluation and in situ localization of apoptosis in fresh and cryopreserved ovarian tissue.Crossref | GoogleScholarGoogle Scholar |
Carvalho, A. A., Faustino, L. R., Silva, C. M., Castro, S. V., Lopes, C. A. P., Santos, R. R., Báo, S. N., Figueiredo, J. R., and Rodrigues, A. P. R. (2013). Novel wide-capacity method for vitrification of caprine ovaries: ovarian tissue cryosytesm (OTC). Anim. Reprod. Sci. 138, 220–227.
| Novel wide-capacity method for vitrification of caprine ovaries: ovarian tissue cryosytesm (OTC).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXksFCjtLg%3D&md5=02df659f15ffbcd56d57b4a420715cdfCAS |
Castelo, T. S., Silva, A. M., Bezerra, L. G. P., Costa, C. Y. M., Lago, A. E. A., Bezerra, J. A. B., Campos, L. B., Praxedes, E. C. G., and Silva, A. R. (2015). Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina). Cryobiology 71, 442–447.
| Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXhsFyhsbvK&md5=e4e8ed4b526a285a8c732fa7fbdc3467CAS |
Castro, S. V., Carvalho, A. A., Silva, C. M. G., Santos, F. W., Campello, C. C., Figueiredo, J. R., and Rodrigues, A. P. R. (2014). Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles. Biopreserv. Biobank. 12, 317–324.
| Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhvVWisLvP&md5=ab9cf5c464a0d0f502f8cdb63c234142CAS |
Cox, S. L., Shaw, J., and Jenkin, G. (1996). Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice. J. Reprod. Fertil. 107, 315–322.
| Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XmsFGgt70%3D&md5=0f10d9f7eb3fe551cb22fa469f4816e7CAS |
Damásio, L. C., Soares-Júnior, J. M., Iavelberg, J., Maciel, G. A. R., Simões, M. J., Simões, R. S., Motta, E. V., Baracat, M. C. P., and Edmund, C. (2016). Heterotopic ovarian transplantation results in less apoptosis than orthotopic transplantation in a minipig model. J. Ovarian Res. 9, 14.
| Heterotopic ovarian transplantation results in less apoptosis than orthotopic transplantation in a minipig model.Crossref | GoogleScholarGoogle Scholar |
Damous, L. L., Nakamuta, J. S., Carvalho, A. E. T. S., Soares, J. M., Simões, M. J., Krieger, J. E., and Baracat, E. C. (2015). Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts. Stem Cell Res. Ther. 6, 57.
| Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts.Crossref | GoogleScholarGoogle Scholar |
Dittrich, R., Lotz, L., Fehm, T., Krüssel, J., von Wolff, M., Toth, B., van der Ven, H., Schüring, A. N., Würfel, W., Hoffmann, I., and Beckmann, M. W. (2015). Xenotransplantation of cryopreserved human ovarian tissue – a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment. Fertil. Steril. 103, 1557–1565.
| Xenotransplantation of cryopreserved human ovarian tissue – a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.Crossref | GoogleScholarGoogle Scholar |
Donnez, J., and Dolmans, M.-M. (2015). Ovarian tissue freezing: current status. Curr. Opin. Obstet. Gynecol. 27, 222–230.
| Ovarian tissue freezing: current status.Crossref | GoogleScholarGoogle Scholar |
Fabbri, R., Vicenti, R., Macciocca, M., Pasquinelli, G., Paradisi, R., Battaglia, C., Martino, N. A., and Venturoli, S. (2014). Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification. BioMed Res. Int. 2014, 673537.
| Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification.Crossref | GoogleScholarGoogle Scholar |
Faustino, L. R., Silva, C. M. G., Rossetto, R., Rodrigues, G. Q., Figueiredo, J. R., and Rodrigues, A. P. R. (2011). Estado atual e desafios da criopreservação de tecido ovariano em mamíferos. Rev. Bras. Reprod. Anim., Belo Horizonte 35, 3–15.
Fontana, C. S., Bencke, G. A., and Reis, R. E. (2003). ‘Livro vermelho de fauna ameaçada de extinção no Rio Grande do Sul.’ (EDIPUCRS: Porto Alegre.)
González, C., Boada, M., Devesa, M., and Veiga, A. (2012). Concise review: fertility preservation: an update. Stem Cells Transl. Med. 1, 668–672.
| Concise review: fertility preservation: an update.Crossref | GoogleScholarGoogle Scholar |
Hemadi, M., Abolhassani, F., Akbari, M., Sobhani, A., Pasbakhsh, P., Ährlund-Richter, L., Modaresi, M. H., and Salehnia, M. (2009). Melatonin promotes the cumulus–oocyte complexes quality of vitrified–thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation. Eur. J. Pharmacol. 618, 84–90.
| Melatonin promotes the cumulus–oocyte complexes quality of vitrified–thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVKkt7fE&md5=3dfd51f0a532f90189bc0692bfbf45b1CAS |
Herraiz, S., Novella-Maestre, E., Rodríguez, B., Díaz, C., Sánchez-Serrano, M., and Pellicer, V. M. A. (2014). Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices. Fertil. Steril. 101, 775–784.e1.
| Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXitVWjtbjF&md5=fef796cd399c3e389bc3cac238e9b66bCAS |
Hoff, J. (2000). Methods of blood collection in the mouse. Lab. Anim. 29, 47–53.
Huang, K. Y., Groot, S. A., Woelders, H., Van Der Horst, G. T. J., Themmene, A. P. N., Colenbrandera, B., and Van Vlissingenb, J. M. F. (2010). Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation. Cryobiology 60, 129–137.
| Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation.Crossref | GoogleScholarGoogle Scholar |
Israely, T., Dafni, H., Granot, D., Nevo, N., Tsafriri, A., and Neeman, M. (2003). Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts. Biol. Reprod. 68, 2055–2064.
| Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXks1GgsL4%3D&md5=776f1b89d755fd64a0b7af70b4c3193dCAS |
International Union for Conservation of Nature (IUCN) (2017). IUCN red list categories and criteria, version 3.1. Available at www.iucnredlist.org [verified 20 January 2017].
Kaneko, H., Nakai, M., Tanihara, F., Noguchi, J., and Kikuchi, K. (2013). Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes. Theriogenology 80, 887–892.
| Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes.Crossref | GoogleScholarGoogle Scholar |
Laschke, M. W., Menger, M. D., and Vollmar, B. (2003). Cryopreservation does not affect neovascularization of freely transplanted ovarian follicles. Fertil. Steril. 79, 1458–1460.
| Cryopreservation does not affect neovascularization of freely transplanted ovarian follicles.Crossref | GoogleScholarGoogle Scholar |
Lee, J., Kim, S. K., Youm, H. W., Kim, H. J., Lee, J. R., Suh, C. S., and Kim, S. H. (2015). Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation. PLoS One 10, e0126252.
| Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.Crossref | GoogleScholarGoogle Scholar |
Lopes, C. A. P., Santos, R. R., Celestino, J. J. H., Melo, M. A., Chaves, R. N., Campello, C. C., Silva, J. R. V., Báo, S. N., Jewgenow, K., and Figueiredo, J. R. (2009). Short- term preservation of canine preantral follicles: effects of temperature, medium and time. Anim. Reprod. Sci. 115, 201–214.
| Short- term preservation of canine preantral follicles: effects of temperature, medium and time.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptl2iurY%3D&md5=0bcb4802f28cfa62d57dd8d431a5f38aCAS |
Motohashi, H. H., and Ishibashi, H. (2016). Cryopreservation of ovaries from neonatal marmoset monkeys. Exp. Anim. 65, 189–196.
| Cryopreservation of ovaries from neonatal marmoset monkeys.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2sXhtFOqtbo%3D&md5=7360b5af5d3cbfb12ba5ecc830b0f331CAS |
Nelson, J. F., Felicio, L. S., Randall, P. K., Sims, C., and Finch, C. E. (1982). A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology. Biol. Reprod. 27, 327–339.
| A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL3s%2FitlCgtg%3D%3D&md5=b5c44feaed15c5c45345e7ad771c0cf9CAS |
Nogueira, S. S. C., and Nogueira-Filho, S. L. G. (2011). Wildlife farming: an alternative to unsustainable hunting and deforestation in Neotropical forests? Biodivers. Conserv. 1, 1–13.
| Wildlife farming: an alternative to unsustainable hunting and deforestation in Neotropical forests?Crossref | GoogleScholarGoogle Scholar |
Nugent, D., Meirow, D., Brook, P. F., Aubard, Y., and Gosden, R. G. (1997). Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum. Reprod. Update 3, 267–280.
| Transplantation in reproductive medicine: previous experience, present knowledge and future prospects.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2svmtleiug%3D%3D&md5=5db8b8d2a987733c738639dc89e7cb38CAS |
Santos, F. A., Pimentel, M. M. L., Teixeira, A. C. G., Cosmo, Í. C., Lima, M. A., Brito, P. D., Macedo, M. F., and Bezerra, M. B. (2016). Follicular right shift: xenografting queens’ ovarian tissue into severe combined imunnodeficiency mice and its responses to exogenous gonadotropin. Theriogenology 86, 1811–1817.
| Follicular right shift: xenografting queens’ ovarian tissue into severe combined imunnodeficiency mice and its responses to exogenous gonadotropin.Crossref | GoogleScholarGoogle Scholar |
Scalercio, S. R. R. A., Brito, A. B., Domingues, S. F. S., Santos, R. R., and Amorim, C. A. (2015). Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi). Reprod. Sci. 22, 68–74.
| Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXitVWmu7g%3D&md5=682781ce77243c21f023c339d6b5c128CAS |
Segino, M., Ikeda, M., Hirahara, F., and Sato, K. (2005). In vitro development of cryopreserved mouse ovarian tissue. Reproduction 130, 187–192.
| In vitro development of cryopreserved mouse ovarian tissue.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVSksbvP&md5=ed06d71cb6cf78a90a757fefd2779046CAS |
Snow, M., Cleary, M., Cox, S. L., Shaw, J. M., Paris, M. C. J., and Jenkin, G. (2002). Generation of live young from xenografted mouse ovaries. Science 297, 2227.
| Generation of live young from xenografted mouse ovaries.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XnvFKqs7c%3D&md5=474d9f7ac18ed29a2396b628ce2b088cCAS |
Stoops, M. A., O’brien, J. K., and Roth, T. L. (2001). Comparison of the effects of in vitro and in situ storage on the viability of mouse ovarian tissue collected after death. Reprod. Fertil. Dev. 13, 389–399.
| Comparison of the effects of in vitro and in situ storage on the viability of mouse ovarian tissue collected after death.Crossref | GoogleScholarGoogle Scholar |
Tahaei, L. S., Eimani, H., Hajmusa, G., Fathi, R., Rezazadeh, V. M., Shahverdi, A., and Eftekhari-Yazdi, P. (2015). Follicle development of xenotransplanted sheep ovarian tissue into male and female immunodeficient rats. Int. J. Fertil. Steril. 9, 354–360.
| 1:CAS:528:DC%2BC2sXmslersL8%3D&md5=4a6e412b70c37de0367a82868fe9d94aCAS |
Von Schonfeldt, V., Chandolia, R., Kiesel, L., Nieschlag, E., Schlatt, S., and Sonntag, B. (2011). Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant. Reproduction 141, 481–490.
| Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3M3nslWquw%3D%3D&md5=cd6f82be772f80ac19b9edefd890d2c8CAS |
Wiedemann, C., Hribal, R., Ringleb, J., Bertelsen, M. F., Rasmusen, K., Andersen, C. Y., Kristensen, S. G., and Jewgenow, K. (2012). Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse. Reprod. Domest. Anim. 47, 300–304.
| Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse.Crossref | GoogleScholarGoogle Scholar |
Wolvekamp, M. C., Cleary, M. L., Cox, S. L., Shaw, J. M., Jenkin, G., and Trounson, A. O. (2001). Follicular development in cryopreserved common wombat ovarian tissue xenografted to nude rats. Anim. Reprod. Sci. 65, 135–147.
| Follicular development in cryopreserved common wombat ovarian tissue xenografted to nude rats.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXht12gs7o%3D&md5=21ce9379f9e38e668c43abaeb707d15fCAS |
Youm, H. W., Lee, J. R., Lee, J., Jee, B. C., Suh, C. S., and Kim, S. H. (2014). Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified–warmed ovarian tissue survival. Hum. Reprod. 29, 720–730.
| Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified–warmed ovarian tissue survival.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXktl2gsbo%3D&md5=a033c511b0be6faed5573945eba507bbCAS |
Zhang, J. M., Li, L. X., Liu, X. L., Yang, Y. X., and Wan, X. P. (2009). Sucrose affecting successful transplantation of vitrified–thawed mouse ovarian tissues. J. Assist. Reprod. Genet. 26, 137–142.
| Sucrose affecting successful transplantation of vitrified–thawed mouse ovarian tissues.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXls1Cgurg%3D&md5=d9a4061bdd53232a11a176b68e819bd6CAS |
Zhou, X. H., Wu, Y. J., Shi, J., Xia, Y. X., and Zheng, S. S. (2010). Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology 60, 101–105.
| Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXivFemt74%3D&md5=6d3a4fab52d7e332cb90feccf08d6ce6CAS |