Modulatory Effects of Single and Complex Vitamins on the In Vitro Growth of Murine Ovarian Follicles

  • Yoon Young Kim
  • Yong Jin Kim
  • Hoon Kim
  • Byeong Cheol KangEmail author
  • Seung-Yup KuEmail author
  • Chang Suk Suh
Original Article



Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated.


In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C + vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining.


Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage.


Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.


Vitamins Murine follicle In vitro maturation L-ascorbic acid 



The authors would like to express sincere thanks to the technical assistance of Mi Ae Lee, Da Young Song and Se Hoon Park. This research was supported by the grants of Ildong Pharmaceutical Co. Ltd (0620154170), Ministry of Science and ICT (2016R1E1A1A01943455) and Seoul National University Hospital (0320170280), Republic of Korea.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The protocols for animal experiments were previewed and approved by the IACUC of Seoul National University Hospital (IACUC No. 18-0029-S1A0) and all experimental procedures were performed under the regulation of Seoul National University Hospital, Department of Experimental Animal Research Unit.


  1. 1.
    Markström E, Svensson ECh, Shao R, Svanberg B, Billig H. Survival factors regulating ovarian apoptosis—dependence on follicle differentiation. Reproduction. 2002;123:23–30.CrossRefGoogle Scholar
  2. 2.
    Kim YJ, Ku SY, Kim YY, Liu HC, Chi SW, Kim SH, et al. MicroRNAs transfected into granulosa cells may regulate oocyte meiotic competence during in vitro maturation of mouse follicles. Hum Reprod. 2013;28:3050–61.CrossRefGoogle Scholar
  3. 3.
    Espey LL. The distribution of collagenous connective tissue in rat ovarian follicles. Biol Reprod. 1976;14:502–6.CrossRefGoogle Scholar
  4. 4.
    Park KE, Ku SY, Jung KC, Liu HC, Kim YY, Kim YJ, et al. Effects of urinary and recombinant gonadotropins on in vitro maturation outcomes of mouse preantral follicles. Reprod Sci. 2013;20:909–16.CrossRefGoogle Scholar
  5. 5.
    Kim YJ, Kim YY, Kang BC, Kim MS, Ko IK, Liu HC, et al. Induction of multiple ovulation via modulation of angiotensin II receptors in in vitro ovarian follicle culture models. J Tissue Eng Regen Med. 2017;11:3100–10.CrossRefGoogle Scholar
  6. 6.
    Kim YY, Kim WO, Liu HC, Rosenwaks Z, Kim JW, Ku SY. Effects of paclitaxel and cisplatin on in vitro ovarian follicle development. Arch Med Sci. 2019. Scholar
  7. 7.
    Thomas FH, Leask R, Srsen V, Riley SC, Spears N, Telfer EE. Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture. Reproduction. 2001;122:487–95.CrossRefGoogle Scholar
  8. 8.
    Rossetto R, Lima-Verde IB, Matos MH, Saraiva MV, Martins FS, Faustino LR, et al. Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles. Domest Anim Endocrinol. 2009;37:112–23.CrossRefGoogle Scholar
  9. 9.
    Gaskins AJ, Chiu YH, Williams PL, Ford JB, Toth TL, Hauser R, et al. Association between serum folate and vitamin B-12 and outcomes of assisted reproductive technologies. Am J Clin Nutr. 2015;102:943–50.CrossRefGoogle Scholar
  10. 10.
    Ranjan R, Ranjan A, Dhaliwal GS, Patra RC. l-Ascorbic acid (vitamin C) supplementation to optimize health and reproduction in cattle. Vet Q. 2012;32:145–50.CrossRefGoogle Scholar
  11. 11.
    Allison RD, Laven RA. Effect of vitamin E supplementation on the health and fertility of dairy cows: a review. Vet Rec. 2000;147:703–8.Google Scholar
  12. 12.
    Murray AA, Molinek MD, Baker SJ, Kojima FN, Smith MF, Hillier SG, et al. Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro. Reproduction. 2001;121:89–96.CrossRefGoogle Scholar
  13. 13.
    Kim YJ, Ku SY, Rosenwaks Z, Liu HC, Chi SW, Kang JS, et al. MicroRNA expression profiles are altered by gonadotropins and vitamin C status during in vitro follicular growth. Reprod Sci. 2010;17:1081–9.CrossRefGoogle Scholar
  14. 14.
    Soleimani Mehranjani M, Mansoori T. Stereological study on the effect of vitamin C in preventing the adverse effects of bisphenol a on rat ovary. Int J Reprod Biomed (Yazd). 2016;14:403–10.CrossRefGoogle Scholar
  15. 15.
    Dumesic DA, Meldrum DR, Katz-Jaffe MG, Krisher RL, Schoolcraft WB. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril. 2015;103:303–16.CrossRefGoogle Scholar
  16. 16.
    Matsuda F, Inoue N, Manabe N, Ohkura S. Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells. J Reprod Dev. 2012;58:44–50.CrossRefGoogle Scholar
  17. 17.
    Gao F, Zhang J, Wang X, Yang J, Chen D, Huff V, et al. Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation. Hum Mol Genet. 2014;23:333–41.CrossRefGoogle Scholar
  18. 18.
    Lohr M, Kaltner H, Lensch M, André S, Sinowatz F, Gabius HJ. Cell-type-specific expression of murine multifunctional galectin-3 and its association with follicular atresia/luteolysis in contrast to pro-apoptotic galectins-1 and -7. Histochem Cell Biol. 2008;130:567–81.CrossRefGoogle Scholar
  19. 19.
    Kim YJ, Park KE, Kim YY, Kim H, Ku SY, Suh CS, et al. Effects of estradiol on the paracrine regulator expression of in vitro maturated murine ovarian follicles. Tissue Eng Regen Med. 2017;14:31–8.CrossRefGoogle Scholar
  20. 20.
    Kim YY, Kim YJ, Cho KM, Kim SH, Park KE, Kang BC, et al. The expression profile of angiotensin system on thawed murine ovaries. Tissue Eng Regen Med. 2016;13:724–31.CrossRefGoogle Scholar
  21. 21.
    Kim YY, Tamadon A, Ku SY. Potential use of antiapoptotic proteins and noncoding RNAs for efficient in vitro follicular maturation and ovarian bioengineering. Tissue Eng Part B Rev. 2017;23:142–58.CrossRefGoogle Scholar
  22. 22.
    Park KE, Kim YY, Ku SY, Baek SM, Huh Y, Kim YJ, et al. Effects of alginate hydrogels on in vitro maturation outcome of mouse preantral follicles. Tissue Eng Regen Med. 2012;9:170–4.CrossRefGoogle Scholar
  23. 23.
    Kim YJ, Ku SY, Jee BC, Suh CS, Kim SH, Choi YM, et al. A comparative study on the outcomes of in vitro fertilization between women with polycystic ovary syndrome and those with sonographic polycystic ovary-only in GnRH antagonist cycles. Arch Gynecol Obstet. 2010;282:199–205.CrossRefGoogle Scholar
  24. 24.
    Paszkowski T, Clarke RN. The Graafian follicle is a site of L-ascorbate accumulation. J Assist Reprod Genet. 1999;16:41–5.CrossRefGoogle Scholar
  25. 25.
    Kuo SM, Lin CP. 17beta-estradiol inhibition of ascorbic acid accumulation in human intestinal Caco-2 cells. Eur J Pharmacol. 1998;361:253–9.CrossRefGoogle Scholar
  26. 26.
    Luck MR, Jeyaseelan I, Scholes RA. Ascorbic acid and fertility. Biol Reprod. 1995;52:262–6.CrossRefGoogle Scholar
  27. 27.
    Huang M, Li J, Teoh H, Man RY. Low concentrations of 17beta-estradiol reduce oxidative modification of low-density lipoproteins in the presence of vitamin C and vitamin E. Free Radic Biol Med. 1999;27:438–41.CrossRefGoogle Scholar
  28. 28.
    Meur SK, Sanwal PC, Yadav MC. Ascorbic acid in buffalo ovary in relation to oestrous cycle. Indian J Biochem Biophys. 1999;36:134–5.Google Scholar
  29. 29.
    Kim YY, Yun JW, Kim JM, Park CG, Rosenwaks Z, Liu HC, et al. Gonadotropin ratio affects the in vitro growth of rhesus ovarian preantral follicles. J Investig Med. 2016;64:888–93.CrossRefGoogle Scholar
  30. 30.
    Donnez J, Dolmans MM. Fertility preservation in women. Nat Rev Endocrinol. 2013;9:735–49.CrossRefGoogle Scholar
  31. 31.
    Speroff L. The effect of aging on fertility. Curr Opin Obstet Gynecol. 1994;6:115–20.CrossRefGoogle Scholar
  32. 32.
    Kim YY, Ku SY, Jang J, Oh SK, Kim HS, Kim SH, et al. Use of long-term cultured embryoid bodies may enhance cardiomyocyte differentiation by BMP2. Yonsei Med J. 2008;49:819–27.CrossRefGoogle Scholar
  33. 33.
    Kim JJ, Choi YM, Chae SJ, Hwang KR, Yoon SH, Kim MJ, et al. Vitamin D deficiency in women with polycystic ovary syndrome. Clin Exp Reprod Med. 2014;41:80–5.CrossRefGoogle Scholar
  34. 34.
    Kim YY, Min H, Kim H, Choi YM, Liu HC, Ku SY. Differential MicroRNA expression profile of human embryonic stem cell-derived cardiac lineage cells. Tissue Eng Regen Med. 2017;14:163–9.CrossRefGoogle Scholar
  35. 35.
    Lee SH, Lee S, Jun HS, Jeong HJ, Cha WT, Cho YS, et al. Expression of the mitochondrial ATPase6 gene and Tfam in down syndrome. Mol Cells. 2003;15:181–5.Google Scholar
  36. 36.
    Ku SY, Suh CS, Kim SH, Choi YM, Kim JG, Moon SY. A pilot study of the use of low dose human menopausal gonadotropin in ovulation induction. Eur J Obstet Gynecol Reprod Biol. 2003;109:55–9.CrossRefGoogle Scholar
  37. 37.
    Kim YJ, Ku SY, Jee BC, Suh CS, Kim SH, Choi YM, et al. A comparative study on the outcomes of in vitro fertilization between women with polycystic ovary syndrome and those with sonographic polycystic ovary-only in GnRH antagonist cycles. Arch Gynecol Obstet. 2010;282:199–205.CrossRefGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society 2019

Authors and Affiliations

  1. 1.Department of Obstetrics and GynecologySeoul National University College of MedicineSeoulRepublic of Korea
  2. 2.Department of Obstetrics and GynecologyKorea University Guro HospitalSeoulRepublic of Korea
  3. 3.Biomedical Research InstituteSeoul National University HospitalSeoulRepublic of Korea

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