ABB  Vol.3 No.1 , February 2012
A modified protocol for in vitro maturation of mouse oocytes from secondary preantral follicles
Abstract: A 2-step culture system was designed and tested for the in vitro maturation efficiency of oocytes from pre-puberty preantral follicles of FVB/N inbred mice. The following modifications were made: 1) The concentration of ITS was reduced by half in the basal MIF medium to minimize uncoordinated growth between oocyte and GC cells; 2) Heterogeneous preantral follicles were cultured in groups of 3 - 5 follicles in hanging drops of medium with reduced concentration of ITS for six days to induction follicular aggregation. This hanging drop method mimics a 3-D IVM culture system at the early stage of cultivation in which the sphere structure of each follicle is well maintained. It also enables follicles in each aggregate to communicate with each other, synchronize their growth, and thus prevent immature follicular rupture. 3) Medium was further supplemented with retinoic acid to enhance developmental capacity of meiotically arrested oocytes. After a 14-day culture in vitro, ~37% of the collected inbred preantral follicles completed nuclear maturation. Approximately 94% of the mature oocytes tested were able to be fertilized; and 77% of them developed into healthy embryos. These results demonstrate that our IVM system is reliable to produce a satisfactory number of high quality oocytes. In addition, multiple cytoplasmic parameters, including gene expression of key regulators, chromosome/spindle organization, mitochondrial proliferation and distribution, and total ATP content were explored to characterize the supportive and limiting components of our IVM system so that the culture system can be further optimized.
Cite this paper: Wang, W. , Tang, Y. , Ni, L. , Jongwutiwes, T. , Liu, H. and Rosenwaks, Z. (2012) A modified protocol for in vitro maturation of mouse oocytes from secondary preantral follicles. Advances in Bioscience and Biotechnology, 3, 57-74. doi: 10.4236/abb.2012.31010.

[1]   Horwath, D., et al. (2007) Subsequent therapeutic options and outcome in couples who fail to fertilize despite in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Clinical & Experimental Obstetrics & Gynecology, 34, 109-110.

[2]   Singh, L.K., Davies, M. and Chatterjee, R. (2005) Fertility in female cancer survivors: Pathophysiology, preservation and the role of ovarian reserve testing. Human Reproduction Update, 11, 69-89. doi:10.1093/humupd/dmh052

[3]   Suikkari, A.M. and V. Soderstrom-Anttila (2007) In-vitro maturation of eggs: Is it really useful? Best Practice & Research Clinical Obstetrics & Gynaecology, 21, 145- 155. doi:10.1016/j.bpobgyn.2006.09.003

[4]   Cha, K.Y., et al. (2005) Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization-embryo transfer. Fertility and Sterility, 83, 1461-1465. doi:10.1016/j.fertnstert.2004.11.044

[5]   Gilchrist, R.B. and J.G. Thompson (2007) Oocyte maturation: Emerging concepts and technologies to improve developmental potential in vitro. Theriogenology, 67, 6-15. doi:10.1016/j.theriogenology.2006.09.027

[6]   Hashimoto, S. (2009) Application of in vitro maturation to assisted reproductive technology. Journal of Reproduction and Development, 55, 1-10. doi:10.1262/jrd.20127

[7]   Eppig, J.J. and Schroeder, A.C. (1989) Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biology of Reproduction, 41, 268-276. doi:10.1095/biolreprod41.2.268

[8]   Cortvrindt, R., Smitz, J. and Van Steirteghem, A.C. (1996) In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Human Reproduction, 11, 2656-2666.

[9]   Xu, M., et al. (2006) Tissue-engineered follicles produce live, fertile offspring. Tissue Engineering, 12, 2739-2746. doi:10.1089/ten.2006.12.2739

[10]   Desai, N., et al. (2010) Three-dimensional in vitro follicle growth: Overview of culture models, biomaterials, design parameters and future directions. Reproductive Biology and Endocrinology, 8, 119. doi:10.1186/1477-7827-8-119

[11]   Nayudu, P.L. and Osborn, S.M. (1992) Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. Journal of Reproduction and Fertility, 95, 349-362. doi:10.1530/jrf.0.0950349

[12]   Boland, N.I., et al. (1993) Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro. Biology of Reproduction, 48, 798-806. doi:10.1095/biolreprod48.4.798

[13]   Xu, M., et al. (2009) Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles. Biology of Reproduction, 81, 587-594. doi:10.1095/biolreprod.108.074732

[14]   Eppig, J.J. (1991) Intercommunication between mammalian oocytes and companion somatic cells. Bioessays, 13, 569- 574. doi:10.1002/bies.950131105

[15]   Carabatsos, M.J., et al. (2000) Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Developmental Biology, 226, 167-179. doi:10.1006/dbio.2000.9863

[16]   Albertini, D.F., et al. (2001) Cellular basis for paracrine regulation of ovarian follicle development. Reproduction, 121, 647-653. doi:10.1530/rep.0.1210647

[17]   McGee, E.A. and Hsueh, A.J. (2000) Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews, 21, 200-214. doi:10.1210/er.21.2.200

[18]   McNatty, K.P., et al. (2007) Control of ovarian follicular development to the gonadotrophin-dependent phase: A 2006 perspective. Society for Reproduction and Fertility Suppleent, 64, 55-68.

[19]   Craig, J., et al. (2007) Gonadotropin and intra-ovarian signals regulating follicle development and atresia: The delicate balance between life and death. Front Biosci, 12, 3628-3639. doi:10.2741/2339

[20]   Kumar, T.R., et al. (1997) Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genetics, 15, 201-204. doi:10.1038/ng0297-201

[21]   Hirshfield, A.N. (1991) Development of follicles in the mammalian ovary. International Review of Cytology, 124, 43-101. doi:10.1016/S0074-7696(08)61524-7

[22]   Gilchrist, R.B., Ritter, L.J. and Armstrong, D.T. (2004) Oocyte-somatic cell interactions during follicle development in mammals. Animal Reproduction Science, 82-83, 431-446. doi:10.1016/j.anireprosci.2004.05.017

[23]   Oktem, O. and Urman, B. (2010) Understanding follicle growth in vivo. Human Reproduction, 25, 2944-2954. doi:10.1093/humrep/deq275

[24]   Eppig, J.J., et al. (1998) Factors affecting the developmental competence of mouse oocytes grown in vitro: Follicle-stimulating hormone and insulin. Biology of Reproduction, 59, 1445-1453. doi:10.1095/biolreprod59.6.1445

[25]   Sanfins, A., et al. (2003) Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes. Biology of Reproduction, 69, 2059-2067. doi:10.1095/biolreprod.103.020537

[26]   Sanfins, A., et al. (2004) Meiotic spindle morphogenesis in in vivo and in vitro matured mouse oocytes: Insights into the relationship between nuclear and cytoplasmic quality. Human Reproduction, 19, 2889-2899. doi:10.1093/humrep/deh528

[27]   Erickson, G.F. and Sorensen, R.A. (1974) In vitro maturation of mouse oocytes isolated from late, middle, and pre-antral graafian follicles. Journal of Experimental Zoology, 190, 123-127. doi:10.1002/jez.1401900112

[28]   Sorensen, R.A. and Wassarman, P.M. (1976) Relationship between growth and meiotic maturation of the mouse oocyte. Developmental Biology, 50, 531-536. doi:10.1016/0012-1606(76)90172-X

[29]   O'Brien, M.J., Pendola, J.K. and Eppig, J.J. (2003) A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biology of Reproduction, 68, 1682-1686. doi:10.1095/biolreprod.102.013029

[30]   Adam, A.A., et al. (2004) In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes. Journal of Reproduction and Development, 50, 579-586. doi:10.1262/jrd.50.579

[31]   De Sousa, P.A., et al. (1998) Temporal patterns of embryonic gene expression and their dependence on oogenetic factors. Theriogenology, 49, 115-128. doi:10.1016/S0093-691X(97)00406-8

[32]   De Vant’ery, C., et al. (1996) An accumulation of p34cdc2 at the end of mouse oocyte growth correlates with the acquisition of meiotic competence. Developmental Biology, 174, 335-344. doi:10.1006/dbio.1996.0078

[33]   Holt, J.E., et al. (2011) The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes. Development, 138, 905- 913. doi:10.1242/dev.059022

[34]   Eppig, J.J. and Wigglesworth, K. (1994) Atypical maturation of oocytes of strain I/LnJ mice. Human Reproduction, 9, 1136-1142.

[35]   Silver, L. (1995) Reproduction and breeding. Mouse genetics, concepts and applications. Oxford University Press, New York, 62-63.

[36]   Kaleta, E. (1977) Influence of genetic factors on the fertilization of mouse ova in vitro. Journal of Reproduction & Fertility, 51, 375-381. doi:10.1530/jrf.0.0510375

[37]   Luckett, D.C. and Mukherjee, A.B. (1986) Embryonic characteristics in superovulated mouse strains. Comparative analyses of the incidence of chromosomal aberrations, morphological malformations, and mortality of embryos from two strains of superovulated mice. Journal of Heredity, 77, 39-42.

[38]   Gao, S., et al. (2004) Genetic variation in oocyte phenotype revealed through parthenogenesis and cloning: Correlation with differences in pronuclear epigenetic modification. Biology of Reproduction, 70, 1162-1170. doi:10.1095/biolreprod.103.024216

[39]   Ibanez, E., Albertini, D.F. and Overstrom, E.W. (2005) Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes. Reproduction, 129, 27-38. doi:10.1530/rep.1.00452

[40]   Suzuki, O., et al. (1996) Development in vitro of preimplantation embryos from 55 mouse strains. Reproduction, Fertility and Development, 8, 975-980. doi:10.1530/rep.1.00452

[41]   Spearow, J.L., et al. (1999) Genetic variation in susceptibility to endocrine disruption by estrogen in mice. Science, 285, 1259-1261. doi:10.1126/science.285.5431.1259

[42]   Polanski, Z. (1997) Genetic background of the differences in timing of meiotic maturation in mouse oocytes: a study using recombinant inbred strains. Journal of Reproduction & Fertility, 109, 109-114. doi:10.1530/jrf.0.1090109

[43]   Wang, W. and Lufkin, T. (2000) The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Developmental Biology, 227, 432-449. doi:10.1006/dbio.2000.9902

[44]   Wang, W., et al. (2001) Hmx2 homeobox gene control of murine vestibular morphogenesis. Development, 128, 5017-5029.

[45]   Kelley, K.A., (2010) Enhancement of IVF in the mouse by zona-drilling. Methods in Enzymology, 476, 229-250. doi:10.1016/S0076-6879(10)76013-4

[46]   Alminana, C., et al. (2008) In vitro maturation of porcine oocytes with retinoids improves embryonic development. Reproduction, Fertility and Development, 20, 483-489. doi:10.1071/RD07175

[47]   Ikeda, S., et al. (2005) The roles of vitamin A for cytoplasmic maturation of bovine oocytes. Journal of Reproduction and Development, 51, 23-35. doi:10.1262/jrd.51.23

[48]   Hattori, M.A., Kato, Y. and Fujihara, N. (2002) Retinoic acid suppression of endothelial nitric oxide synthase in porcine oocyte. Canadian Journal of Physiology and Pharmacology, 80, 777-782. doi:10.1139/y02-099

[49]   Tahaei, L.S., et al. (2011) Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system. Journal of Assisted Reproduction and Genetics, 28, 553-558. doi:10.1007/s10815-011-9579-8

[50]   Gilchrist, R.B., et al. (2006) Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation. Journal of Cell Science, 119, 3811-3821. doi:10.1242/jcs.03105

[51]   Orisaka, M., et al. (2009) Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis. Endocrinology, 150, 2740-2748. doi:10.1210/en.2008-1536

[52]   Thomas, F.H., et al. (2001) Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture. Reproduction, 122, 487-495. doi:10.1530/rep.0.1220487

[53]   Fulop, C., et al. (2003) Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development, 130, 2253-2261. doi:10.1242/dev.00422

[54]   Ny, T., Wahlberg, P. and Brandstrom, I.J. (2002) Matrix remodeling in the ovary: Regulation and functional role of the plasminogen activator and matrix metalloproteinase systems. Molecular and Cellular Endocrinology, 187, 29-38. doi:10.1016/S0303-7207(01)00711-0

[55]   Kerr, J.F., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 26, 239-257. doi:10.1038/bjc.1972.33

[56]   Roberts, R., et al. (2005) Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro. Biology of Reproduction, 72, 107-118. doi:10.1095/biolreprod.104.032003

[57]   Combelles, C.M., et al. (2002) Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Human Reproduction, 17, 1006-1016. doi:10.1093/humrep/17.4.1006

[58]   Vanhoutte, L., et al. (2007) Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor. Human Reproduction, 22, 1239-1246. doi:10.1093/humrep/dem007

[59]   Mullen, S.F., Rosenbaum, M. and Critser, J.K. (2007) The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes. Cryobiology, 54, 281-289. doi:10.1016/j.cryobiol.2007.03.005

[60]   Sun, Q.Y., et al. (2001) Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro. Reproduction, 122, 155-163. doi:10.1530/rep.0.1220155

[61]   Adona, P.R., et al. (2008) Prematuration of bovine oocytes with butyrolactone I: Effects on meiosis progression, cytoskeleton, organelle distribution and embryo development. Animal Reproduction Science, 108, 49-65. doi:10.1016/j.anireprosci.2007.07.002

[62]   Wassarman, P.M., et al. (1979) Meiotic maturation of mouse oocytes in vitro. Advances in Experimental Medicine and Biology, 112, 251-268.

[63]   Van Blerkom, J. (1991) Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proceedings of the National Academy of Sciences of the United States of America, 88, 5031-5035. doi:10.1073/pnas.88.11.5031

[64]   Buccione, R., Schroeder, A.C. and Eppig, J.J. (1990) Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biology of Reproduction, 43, 543-547. doi:10.1095/biolreprod43.4.543

[65]   Su, Y.Q., Sugiura, K. and Eppig, J.J. (2009) Mouse oocyte control of granulosa cell development and function: Paracrine regulation of cumulus cell metabolism. Seminars in Reproductive Medicine, 27, 32-42. doi:10.1055/s-0028-1108008

[66]   Vanderstichele, H., et al. (1994) Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines. Biology of Reproduction, 50, 1190-1202. doi:10.1095/biolreprod50.5.1190

[67]   Halliwell, B. and Cross, C.E. (1994) Oxygen-derived species: Their relation to human disease and environmental stress. Environmental Health Perspectives, 102, 5-12.

[68]   Nasr-Esfahani, M.H., Aitken, J.R. and Johnson, M.H. (1990) Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development, 109, 501-507.

[69]   Lopes, S., et al. (1998) Reactive oxygen species: Potential cause for DNA fragmentation in human spermatozoa. Human Reproduction, 13, 896-900. doi:10.1093/humrep/13.4.896

[70]   Kowaltowski, A.J. and Vercesi, A.E. (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biology and Medicine, 26, 463-471. doi:10.1016/S0891-5849(98)00216-0

[71]   Lee, M.S., et al. (2005) The beneficial effects of insulin and metformin on in vitro developmental potential of porcine oocytes and embryos. Biology of Reproduction, 73, 1264-1268. doi:10.1095/biolreprod.105.041186

[72]   Eppig, J.J., et al. (2000) Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid. Molecular and Cellular Endocrinology, 163, 109-116. doi:10.1016/S0303-7207(99)00247-6

[73]   Pickering, S.J., et al. (1988) Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Human Reproduction, 3, 978-989.

[74]   Tao, W., et al. (2005) Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell, 8, 49-59. doi:10.1016/j.ccr.2005.06.003

[75]   Eroglu, A., et al. (1998) Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes. Journal of Assisted Reproduction and Genetics, 15, 447-454. doi:10.1007/BF02744940

[76]   Rienzi, L., et al. (2003) Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Human Reproduction, 18, 1289-1293. doi:10.1093/humrep/deg274

[77]   Rienzi, L., et al. (2005) Meiotic spindle visualization in living human oocytes. Reproductive BioMedicine Online, 10, 192-198. doi:10.1016/S1472-6483(10)60940-6

[78]   Ibanez, E., et al. (2005) Genetic strain variations in the metaphase-II phenotype of mouse oocytes matured in vivo or in vitro. Reproduction, 130, 845-855. doi:10.1530/rep.1.00558

[79]   Guerin, P., El Mouatassim, S. and Menezo, Y. (2001) Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Human Reproduction Update, 7, 175-189. doi:10.1093/humupd/7.2.175

[80]   Ferreira, E.M., et al. (2009) Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence. Theriogenology, 71, 836-848. doi:10.1016/j.theriogenology.2008.10.023

[81]   Gosden, R., Krapez, J. and Briggs, D. (1997) Growth and development of the mammalian oocyte. Bioessays, 19, 875-882. doi:10.1002/bies.950191007

[82]   Van Blerkom, J., Sinclair, J. and Davis, P. (1998) Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy. Human Reproduction, 13, 2857-2868.

[83]   Zeng, H.T., et al. (2007) Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes. Human Reproduction, 22, 1681-1686. doi:10.1093/humrep/dem070

[84]   Takeuchi, T., et al. (2005) Effect of treating induced mitochondrial damage on embryonic development and epigenesis. Biology of Reproduction, 72, 584-592. doi:10.1095/biolreprod.104.032391

[85]   Bavister, B.D. and Squirrell, J.M. (2000) Mitochondrial distribution and function in oocytes and early embryos. Human Reproduction, 15, 189-198.

[86]   El Shourbagy, S.H., et al. (2006) Mitochondria directly influence fertilisation outcome in the pig. Reproduction, 131, 233-245. doi:10.1530/rep.1.00551

[87]   Santos, T.A., El Shourbagy, S. and St. John, J.C. (2006) Mitochondrial content reflects oocyte variability and fertilization outcome. Fertility and Sterility, 85, 584-591. doi:10.1016/j.fertnstert.2005.09.017

[88]   Dumollard, R., et al. (2008) Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes. Developmental Biology, 316, 431-440. doi:10.1016/j.ydbio.2008.02.004

[89]   Wai, T., et al. (2010) The role of mitochondrial DNA copy number in mammalian fertility. Biology of Reproduction, 83, 52-62. doi:10.1095/biolreprod.109.080887

[90]   Reynier, P., et al. (2001) Mitochondrial DNA content affects the fertilizability of human oocytes. Molecular Human Reproduction, 7, 425-429. doi:10.1093/molehr/7.5.425

[91]   Wilding, M., et al. (2001) Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Human Reproduction, 16, 909-917. doi:10.1093/humrep/16.5.909

[92]   Van Blerkom, J., Davis, P. and Alexander, S. (2000) Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: Relationship to microtubular organization, ATP content and competence. Human Reproduction, 15, 2621-2633. doi:10.1093/humrep/15.12.2621

[93]   Ylikallio, E., et al. (2010) High mitochondrial DNA copy number has detrimental effects in mice. Human Molecular Genetics, 19, 2695-2705. doi:10.1093/hmg/ddq163

[94]   Duque, P., et al. (2002) Enhancement of developmental capacity of meiotically inhibited bovine oocytes by retinoic acid. Human Reproduction, 17, 2706-2714. doi:10.1093/humrep/17.10.2706

[95]   Atikuzzaman, M., et al. (2011) The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production. Biology of Reproduction, 84, 1272-1281. doi:10.1095/biolreprod.110.086595