Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

The in vitro Analysis of Quality of Ovarian Follicle Culture Systems Using Time-Lapse Microscopy and Quantitative Real-Time PCR

Authors
Maxim Filatov 1
Denis Nikishin 2
Yulia Khramova 1
Maria Semenova 1
1 Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
2 Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia
Abstract
Background: The aim of ovarian follicle in vitro culture is to obtain mature oocytes. To evaluate the efficiency of in vitro culture system, the status of the cultured oocyte can be analyzed. Methods: The preantral ovarian follicles retrieved from 14-day-old C57Bl/6J mice were cultured in 3D alginate hydrogel. The status of oocytes obtained from mature (3 months old, group A) and immature (3 weeks old, group B) mice was compared to the status of oocytes retrieved from ovarian follicles cultured in vitro (Group C) using qRT-PCR analysis and time-lapse microscopy. In the qRT-PCR analysis, 8 samples for group A (80 oocytes), 8 samples for group B (80 oocytes), and 6 samples for group C (60 oocytes) were included. Time-lapse analysis was performed in group A (oocytes n=31), group B (n=45), and group C (n=21). Statistical analysis was done by Kruskal-Wallis and chi-square tests and differences were considered statistically significant if p<0,05. Results: The diameter of group C oocytes is lower in comparison to group A oocytes (67 μm vs. 75 μm, correspondingly). Groups B and C oocytes exhibited delayed meiosis in comparison to group A oocytes. Expression levels of six oocyte maturation genes (Ccnb, CDK1, Ccnh, Wee2, Mos and Epab) were evaluated using qRT-PCR analysis. Expression levels of Ccnh and Epab are lowered in group C oocytes compared to the expression levels of these genes in groups A and B oocytes (p<0.05). Conclusion: Oocytes obtained after ovarian follicles in vitro culture have reduced development competence, future fundamental changes of in vitro culture systems can be expected.
Keywords
in vitro culture
alginate
Mice
oocyte
Ovarian follicle
real
time PCR
time
lapse microscopy
  1. Filatov MA, Khramova YV, Kiseleva MV, Malinova IV, Komarova EV, Semenova ML. Female fertility preservation strategies: cryopreservation and ovarian tissue in vitro culture, current state of the art and future perspectives. Zygote. 2016;24(5):635-53.
  2. Dunning KR, Akison LK, Russell DL, Norman RJ, Robker RL. Increased beta-oxidation and improved oocyte developmental competence in response to l-carnitine during ovarian in vitro follicle development in mice. Biol Reprod. 2011;85(3):548-55.
  3. Hornick JE, Duncan FE, Shea LD, Woodruff TK. Multiple follicle culture supports primary follicle growth through paracrine-acting signals. Reproduction. 2013;145(1):19-32.
  4. Jiao ZX, Woodruff TK. Follicle microenvironment-associated alterations in gene expression in the mouse oocyte and its polar body. Fertil Steril. 2013;99(5):1453-59.e1.
  5. Gook DA, Edgar DH, Lewis K, Sheedy JR, Gardner DK. Impact of oxygen concentration on adult murine pre-antral follicle development in vitro and the corresponding metabolic profile. Mol Hum Reprod. 2014;20(1):31-41.
  6. Makanji Y, Tagler D, Pahnke J, Shea LD, Woodruff TK. Hypoxia-mediated carbohydrate metabolism and transport promote early-stage murine follicle growth and survival. Am J Physiol Endocrinol Metab. 2014;306(8):E893-903.
  7. Filatov MA, Khramova YV, Semenova ML. In vitro mouse ovarian follicle growth and maturation in alginate hydrogel: current state of the art. Acta Naturae. 2015;7(2):48-56.
  8. Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue-engineered follicles produce live, fertile offspring. Tissue Eng. 2006;12(10):2739-46.
  9. Wang X, Catt S, Pangestu M, Temple-Smith P. Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births. Reproduction. 2011;141(2):183-91.
  10. Mochida N, Akatani-Hasegawa A, Saka K, Ogino M, Hosoda Y, Wada R, et al. Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice. Reproduction. 2013;146(1):37-47.
  11. Higuchi CM, Maeda Y, Horiuchi T, Yamazaki Y. A simplified method for three-dimensional (3-D) ovarian tissue culture yielding oocytes competent to produce full-term offspring in mice. PLoS One. 2015;10(11):e0143114.
  12. Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials. 2006;27(5):714-23.
  13. Xu M, West E, Shea LD, Woodruff TK. Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development. Biol Reprod. 2006;75(6):916-23.
  14. Shikanov A, Xu M, Woodruff TK, Shea LD. Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development. Biomaterials. 2009;30(29):5476-85.
  15. Xu M, Banc A, Woodruff TK, Shea LD. Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles. Biotechnol Bioeng. 2009;103(2):378-86.
  16. Jin SY, Lei L, Shikanov A, Shea LD, Woodruff TK. A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse. Fertil Steril. 2010;93(8):2633-9.
  17. Filatov M, Khramova Y, Semenova M. Molecular mechanisms of prophase I meiotic arrest maintenance and meiotic resumption in mammalian oocytes. Reprod Sci. 2019;26(11):1519-37.
  18. West-Farrell ER, Xu M, Gomberg MA, Chow YH, Woodruff TK, Shea LD. The mouse follicle microenvironment regulates antrum formation and steroid production: alterations in gene expression profiles. Biol Reprod. 2009;80(3):432-9.
  19. Parrish EM, Siletz A, Xu M, Woodruff TK, Shea LD. Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system. Reproduction. 2011;142(2):309-18.
  20. Amoushahi M, Salehnia M, Mowla SJ, Ghorbanmehr N. Morphological and molecular aspects of in vitro culture of preantral follicles derived from vitrified ovarian. Cell J. 2017;19(3):332-42.
  21. Sánchez F, Romero S, Albuz FK, Smitz J. In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence. J Assist Reprod Genet. 2012;29(2):141-52.
  22. Adhikari D, Zheng W, Shen Y, Gorre N, Ning Y, Halet G, et al. Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. Hum Mol Genet. 2012;21(11):2476-84.
  23. Kong BY, Bernhardt ML, Kim AM, O’Halloran TV, Woodruff TK. Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway. Biol Reprod. 2012;87(1):1-12.
  24. Yuan J, Xu BZ, Qi ST, Tong JS, Wei L, Li M, et al. MAPK-activated protein kinase 2 is required for mouse meiotic spindle assembly and kinetochore-microtubule attachment. PLoS One. 2010;5(6):e11247.
  25. Sha QQ, Dai XX, Dang Y, Tang F, Liu J, Zhang YL, et al. A MAPK cascade couples maternal mRNA translation and degradation to meiotic cell cycle progression in mouse oocytes. Development. 2017;144(3):452-63.
  26. Wilkie GS, Gautier P, Lawson D, Gray NK. Embryonic poly(A)-binding protein stimulates translation in germ cells. Mol Cell Biol. 2005;25(5):2060-71.
  27. Guzeloglu-Kayisli O, Lalioti MD, Aydiner F, Sasson I, Ilbay O, Sakkas D, et al. Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice. Biochem J. 2012;446(1):47-58.
  28. Ozturk S, Yaba-Ucar A, Sozen B, Mutlu D, Demir N. Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos. Reprod Fertil Dev. 2016;28(3):375-83.
  29. Faramarzi A, Khalili MA, Agha-Rahimi A, Omidi M. Is there any correlation between oocyte polarization microscopy findings with embryo time lapse monitoring in ICSI program? Arch Gynecol Obstet. 2017;295(6):1515-22.
  30. Truong T, Gardner DK. Antioxidants improve IVF outcome and subsequent embryo development in the mouse. Hum Reprod. 2017;32(12):2404-13.
  31. Barberet J, Bruno C, Valot E, Antunes-Nunes C, Jonval L, Chammas J, et al. Can novel early non-invasive biomarkers of embryo quality be identified with time-lapse imaging to predict live birth? Hum Reprod. 2019;34(8):1439-49.
  32. Cobo A, Coello A, Remohí J, Serrano J, de Los Santos JM, Meseguer M. Effect of oocyte vitrification on embryo quality: time-lapse analysis and morphokinetic evaluation. Fertil Steril. 2017;108(3):491-7.e3.
  33. Belli M, Vigone G, Merico V, Redi CA, Garagna S, Zuccotti M. Time-lapse dynamics of the mouse oocyte chromatin organisation during meiotic resumption. Biomed Res Int. 2014;2014:207357.
  34. Cavalera F, Zanoni M, Merico V, Sacchi L, Bellazzi R, Garagna S, et al. Chromatin organization and timing of polar body I extrusion identify developmentally competent mouse oocytes. Int J Dev Biol. 2019;63(3-4-5):245-51.
  35. Cavalera F, Zanoni M, Merico V, Bui TTH, Belli M, Fassina L, et al. A neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes. J Vis Exp. 2018;(133).
  36. Zenclussen ML, Casalis PA, Jensen F, Woidacki K, Zenclussen AC. Hormonal fluctuations during the estrous cycle modulate heme oxygenase-1 expression in the uterus. Front Endocrinol (Lausanne). 2014;5:32.
  37. Riedel G, Rüdrich U, Fekete-Drimusz N, Manns MP, Vondran FW, Bock M. An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells. PLoS One. 2014;9(3):e93031.
  38. Mehlmann LM. Losing mom's message: requirement for DCP1A and DCP2 in the degradation of maternal transcripts during oocyte maturation. Biol Reprod. 2013;88(1):10.
  39. Phelan JP, Austad SN. Selecting animal models of human aging: inbred strains often exhibit less biological uniformity than F1 hybrids. J Gerontol. 1994;49(1):B1-11.
  40. Hartman JL, Garvik B, Hartwell L. Principles for the buffering of genetic variation. Science. 2001;291(5506):1001-4.
  41. Hartwell L. Genetics. Robust interactions. Science. 2004;303(5659):774-5.
  42. Cui X, Affourtit J, Shockley KR, Woo Y, Churchill GA. Inheritance patterns of transcript levels in F1 hybrid mice. Genetics. 2006;174(2):627-37.
  43. Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ, Robker RL. Beta-oxidation is essential for mouse oocyte developmental competence and early embryo development. Biol Reprod. 2010;83(6):909-18.
  44. Oktem O, Buyuk E, Oktay K. Preantral follicle growth is regulated by c-Jun-N-terminal kinase (JNK) pathway. Reprod Sci. 2011;18(3):269-76.
  45. Kohaya N, Fujiwara K, Ito J, Kashiwazaki N. Generation of live offspring from vitrified mouse oocytes of C57BL/6J strain. PLoS One. 2013;8(3):e58063.
  46. Takeo T, Nakagata N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS One. 2015;10(5):e0128330.
  47. West ER, Xu M, Woodruff TK, Shea LD. Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials. 2007;28(30):4439-48.
  48. Tagler D, Makanji Y, Tu T, Bernabé BP, Lee R, Zhu J, et al. Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels. Biotechnol Bioeng. 2014;111(7):1417-29.
  49. Pretty CG, Le Compte A, Penning S, Fisk L, Shaw GM, Desaive T, et al. Interstitial insulin kinetic parameters for a 2-compartment insulin model with saturable clearance. Comput Methods Programs Biomed. 2014;114(3):e39-45.
  50. Nielsen TK, Højgaard M, Andersen JT, Poulsen HE, Lykkesfeldt J, Mikines KJ. Elimination of ascorbic acid after high-dose infusion in prostate cancer patients: a pharmacokinetic evaluation. Basic Clin Pharmacol Toxicol. 2015;116(4):343-8.
  51. Boyle KL, Leech E. A review of the pharmacology and clinical uses of pimobendan. J Vet Emerg Crit Care (San Antonio). 2012;22(4):398-408.
  52. Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A in rat oocyte maturation. Biol Reprod. 2001;65(5):1444-51.
  53. Rossi DJ, Londesborough A, Korsisaari N, Pihlak A, Lehtonen E, Henkemeyer M, et al. Inability to enter S phase and defective RNA polymerase II CTD phosphorylation in mice lacking Mat1. EMBO J. 2001;20(11):2844-56.
  54. Patel SA, Simon MC. Functional analysis of the Cdk7.cyclin H.Mat1 complex in mouse embryonic stem cells and embryos. J Biol Chem. 2010;285(20):15587-98.
  55. Kolesnikova O, Radu L, Poterszman A. TFIIH: a multi-subunit complex at the cross-roads of transcription and DNA repair. Adv Protein Chem Struct Biol. 2019;115:21-67.
  56. Zhu Q, Wani G, Sharma N, Wani A. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair. DNA Repair (Amst). 2012;11(12):942-50.
  57. Compe E, Drané P, Laurent C, Diderich K, Braun C, Hoeijmakers JH, et al. Dysregulation of the peroxisome proliferator-activated receptor target genes by XPD mutations. Mol Cell Biol. 2005;25(14):6065-76.
  58. Helenius K, Yang Y, Alasaari J, Mäkelä TP. Mat1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipocyte differentiation. Mol Cell Biol. 2009;29(2):315-23.
  59. Kalous J, Kubelka M, Motlík J. The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes. Zygote. 2003;11(1):61-8.
  60. Chang HM, Qiao J, Leung PC. Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors. Hum Reprod Update. 2016;23(1):1-18.
  61. Janas P, Kucybała I, Radoń-Pokracka M, Huras H. Telocytes in the female reproductive system: an overview of up-to-date knowledge. Adv Clin Exp Med. 2018;27(4):559-65.
  62. Tagler D, Tu T, Smith RM, Anderson NR, Tingen CM, Woodruff TK, et al. Embryonic fibroblasts enable the culture of primary ovarian follicles within alginate hydrogels. Tissue Eng Part A. 2012;18(11-12):1229-38.
  63. Choi JK, Agarwal P, He X. In vitro culture of early secondary preantral follicles in hanging drop of ovarian cell-conditioned medium to obtain MII oocytes from outbred deer mice. Tissue Eng Part A. 2013;19(23-24):2626-37.
  64. Choi JK, Agarwal P, Huang H, Zhao S, He X. The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue. Biomaterials. 2014;35(19):5122-8.
  65. Shikanov A, Xu M, Woodruff TK, Shea LD. A method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system. J Vis Exp. 2011;(49). pii: 2695.
Journal of Reproduction & Infertility
Volume 21, Issue 2
Spring 2020
Pages 94-106