Combined space-organized system for animals oocytes maturation in vitro

  • V. J. Syrvatka
  • Yu. I. Slyvchuk
  • O. V. Shtapenko
  • O. M. Gromyko
  • I. I. Gevkan


Aim. The aim of the work was to create a spaсe-organized system for oocytes maturation based on rabbits granulosa cells, hyaluronic acid (for structural organization of this system) and insulin, as an important growth factor. Methods. Cultivation of granulosa cells and oocytes maturation in vitro, biochemical determination of lactate dehydrogenase activity and cholesterol concentration, and determination of progesterone and estradiol concentrations by enzyme immunoassay were done. Results. Hyaluronic acid in concentration of 0.5 μg/ml increased proliferation of granulosa cells and progesterone concentration in the conditioned medium on 72 h of cultivation, and estradiol on 48 and 72 hours of cultivation was found. The degree of oocyte maturation to metaphase-2 was 85% by the influence of 10 μg/ml insulin. Same concentration of insulin increased granulosa cells proliferation, estradiol, progesterone and cholesterol concentration in the conditioning medium after 24 hours of cultivation. Conclusions. The optimal parameters of the combined spaceorganized culture system are 1 million cells/ml of granulose with the addition of 0.5 μg/ml hyaluronic acid and 10 μg/ml insulin, which creates the optimal conditions for the maturation of oocytes to metaphase-2 cell cycle.

Keywords: oocytes, granulosa cells, hyaluronic acid, insulin, in vitro.


Lonergan P., Fair T. Maturation of oocytes in vitro. Annual Review of Animal Biosciences. 2016. Vol. 4. P. 255-268. doi. 10.1146/annurev-animal-022114-110822.

Silva V., van den Hurk R., Figueiredo J.R. Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals. Domest Anim Endocrinol. 2016. Vol. 55. P. 123-135. doi:10.1016/j.domaniend.2015.12.006.

Gilchrist R.B. Recent insights into oocyte-follicle cell interactions provide opportunities for the development of new approaches to in vitro maturation. Reprod Fertil Dev. 2011. Vol. 23. P. 23–31. doi: 10.1071/RD10225.

Jahromi B.N., Mosallanezhad Z., Matloob N., Davari M., Ghobadifar M.A. The potential role of granulosa cells in the maturation rate of immature human oocytes and embryo development: A co-culture study. Clin Exp Reprod Med. 2015 Vol. 42. No. 3. P. 111–117. doi: 10.5653/cerm.2015.42.3.111.

Li Z. Zhang P., Zhang Z. Pan B., Chao H., Li L., Pan Q., Shen W. A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes. Histochemistry & Cell Biology. 2011. Vol. 135, No. 5. P. 513–520. doi: 10.1007/s00418-011-0812-4.

Alam M.H., Miyano T. Interaction between growing oocytes and granulosa cells in vitro. Reproductive Medicine and Biology. 2020. Vol. 19. No. 1. P. 13-23. doi:10.1002/rmb2.12292.

Casper R., Haas J., Hsieh T.-B., Bassil R., Mehta C. Recent advances in in vitro fertilization. Version 1. F1000Res. 2017. Vol. 6. P 1616. doi: 10.12688/f1000research.11701.1.

Mareiab W.F., Ghafaric F., Fouladi-Nashta A.A. Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes. Theriogenology. 2012. Vol. 7, No. 3. P. 670-677. doi:10.1016/j.theriogenology.2012.03.013.

Zhao G., Zhou X., Fang T., Hou Y., Hu Y. Hyaluronic acid promotes the expression of progesterone receptor membrane component 1 via epigenetic silencing of miR-139-5p in human and rat granulosa. Cells Biology of Reproduction. 2014. Vol. 91. No. 5. P. 1-9, doi:10.1095/biolreprod.114.120295

Lackey B., Gray D., Henricks N. Physiological basis for use on IGFs in reproductive application: A Review. Theriogenology. 2000. Vol. 53. №5. P. 1147–1156. doi:10.1016/S0093-691X(00)00259-4.

Riosa G.L., Buschiazzoab J., Muccia N.C., Kaisera G.G., Cesaric A., Alberioa R.H. Combined epidermal growth factor and hyaluronic acid supplementation of in vitro maturation medium and its impact on bovine oocyte proteome and competence. Theriogenology. 2015. Vol. 83. No. 5. P. 874-880 doi:10.1016/j.theriogenology.2014.11.022.