Role of Visfatin in Restoration of Ovarian Aging and Fertility in the Mouse Aged 18 Months

  • Byung-Kyoo Park
  • Min Jung Park
  • Hwi Gon Kim
  • Si Eun Han
  • Chang-Woon Kim
  • Bo Sun JooEmail author
  • Kyu-Sup LeeEmail author
Original Article


The activation of dormant primordial follicles and ovarian angiogenesis has been attempted as a new treatment strategy for age-related ovarian aging. This study examined whether visfatin rescues age-related fertility decline in female mice aged 18 months, and whether this effect relates to the mTOR/PI3K signaling pathways for activation of primordial follicles and ovarian angiogenesis. Female mice were intraperitoneally injected with 0.1 ml of 500 ng/ml or 1000 ng/ml of visfatin three times at intervals of 2 days, and both ovaries were provided for H&E staining. In another experiment, the mice were superovulated with pregnant mare’s serum gonadotropin and human chorionic gonadotropin, and were mated with males. After 18 h, zygotes were collected and cultured for 4 days, and numbers and embryo developmental competency of zygotes retrieved were evaluated. The expression of mTOR/PI3K signaling pathway regulated genes (4EBP1, S6K1, and RPS6) and angiogenic factors (VEGF, visfatin, and SDF-1α) in the ovary were examined. As well, visfatin-treated mice were mated with male mice for 2 weeks, and the pregnancy outcome was monitored up to 3 weeks. Visfatin significantly increased the total numbers of follicles compared with control. Numbers of zygotes retrieved, blastocyst formation rate, and pregnancy rate were significantly increased at 500 ng/ml of visfatin (2.83%, 40.0%, and 80%, respectively) compared with control (0, 0, and no pregnancy). Ovarian expressions of S6K1, RPS6, VEGF, visfatin, and SDF-1α were significantly stimulated at 500 ng/ml of visfatin. These results show that visfatin treatment of an optimal dose rescues age-related decline in fertility, possibly by stimulating mTOR/PI3K signaling.


Visfatin Ovarian aging Fertility Primordial follicle activation mTOR/PI3K 


Funding Information

This study was supported by a grant of the Basic Research Project, Ministry of Science and ICT and National Research Foundation, Republic of Korea (2017R1A2B4010859).

Compliance with Ethical Standards

This study was approved by the institutional review board of Pusan National University Hospital, Korea. All animal experiments were conducted under the guidance for the Care and Use of Laboratory Animals of the National Institutes of Health, approved by the Pusan National University Hospital Institutional Animal Care and Use Committee.

Conflict of Interests

The authors declare that they have no conflict of interest.


  1. 1.
    Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC. Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab. 2007;18:58–65.CrossRefGoogle Scholar
  2. 2.
    Navot D, Drews MR, Bergh PA, Guzman I, Karstaedt A, Scott RT Jr, et al. Age-related decline in female fertility is not due to diminished capacity of the uterus to sustain embryo implantation. Fertil Steril. 1994;61:97–101.CrossRefGoogle Scholar
  3. 3.
    Simpson JL. Lobo RA, Kelsey J, Marcus R, eds. Genetic programming in ovarian development and oogenesis. Menopause: biology and pathobiology. San Diego: Academic Press; 2000: 77–94.Google Scholar
  4. 4.
    Keefe DL, Niven-Fairchild T, Powell S, Buradagunta S. Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women. Fertil Steril. 1995;64:577–83.CrossRefGoogle Scholar
  5. 5.
    Thouas GA, Trounson AO, Jones GM. Effect of female age on mouse oocyte developmental competence following mitochondrial injury. Biol Reprod. 2005;73:366–673.CrossRefGoogle Scholar
  6. 6.
    Bentov Y, Casper RF. The aging oocyte--can mitochondrial function be improved? Fertil Steril. 2013;99:18–22.CrossRefGoogle Scholar
  7. 7.
    te Velde ER, Peasron PL. The variability of female reproductive aging. Hum Reprod Update. 2002;8:141–54.CrossRefGoogle Scholar
  8. 8.
    Tatone C, Amicarelli F, Carbone MC, Monteleone P, Caserta D, Marci R, et al. Cellular and molecular aspects of ovarian follicle ageing. Hum Reprod Update. 2008;14:131–42.CrossRefGoogle Scholar
  9. 9.
    Redmer CA, Reynolds LP. Angiogenesis in the ovary. Rev Reprod. 1996;1:182–92.CrossRefGoogle Scholar
  10. 10.
    Lee DH, Joo BS, Suh DS, Park JH, Choi YM, Lee KS. Sodium nitroprusside treatment during the superovulation process improves ovarian response and ovarian expression of vascular endothelial growth factor in aged female mice. Fertil Steril. 2008;89:1514–21.CrossRefGoogle Scholar
  11. 11.
    Ha CS, Joo BS, Kim SC, Joo JK, Kim HG, Lee KS. Estrogen administration during superovulation increases oocyte quality and expressions of vascular endothelial growth factor and nitric oxide synthase in the ovary. J Obstet Gynaecol Res. 2010;36:789–95.CrossRefGoogle Scholar
  12. 12.
    Choi KH, Joo BS, Sun ST, et al. Administration of visfatin during superovulation improves developmental competency of oocytes and fertility potential in aged female mice. Fertil Steril. 2012;97:1234–41.CrossRefGoogle Scholar
  13. 13.
    Geva E, Jaffe RB. Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril. 2000;74:429–38.CrossRefGoogle Scholar
  14. 14.
    Fraser HM. Regulation of the ovarian follicular vasculature. Reprod Biol Endocrinol. 2006;4:18–26.CrossRefGoogle Scholar
  15. 15.
    McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21:200–14.Google Scholar
  16. 16.
    Adhikari D, Liu K. Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocr Rev. 2009;30:438–64.CrossRefGoogle Scholar
  17. 17.
    Hsueh AJ, Kawamura K, Cheng Y, Fauser BC. Intraovarian control of early folliculogenesis. Endocr Rev. 2015;36:1–24.CrossRefGoogle Scholar
  18. 18.
    Shea LD, Woodruff TK, Shikanov A. Bioengineering the ovarian follicle microenvironment. Annu Rev Biomed Eng. 2014;16:29–52.CrossRefGoogle Scholar
  19. 19.
    Li J, Zhou F, Zheng T, Pan Z, Liang X, Huang J, et al. Ovarian germline stem cells (OGSCs) and the hippo signaling pathway association with physiological and pathological ovarian aging in mice. Cell Physiol Biochem. 2015;36:1712–24.CrossRefGoogle Scholar
  20. 20.
    Celik O, Celik N, Gungor S, Haberal ET, Aydin S. Selective regulation of oocyte meiotic events enhances progress in fertility preservation methods. Biochem Insights. 2015;8:11–21.CrossRefGoogle Scholar
  21. 21.
    Kawamura K, Cheng Y, Suzuki N, Deguchi M, Sato Y, Takae S, et al. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc Natl Acad Sci U S A. 2013;10:17474–9.CrossRefGoogle Scholar
  22. 22.
    Sun X, Su Y, He Y, Zhang J, Liu W, Zhang H, et al. New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators. Cell Cycle. 2015;14:721–31.CrossRefGoogle Scholar
  23. 23.
    Zheng W, Nagaraju G, Liu Z, Liu K. Functional roles of the phosphatidylinositol 3-kinases (PI3Ks) signaling in the mammalian ovary. Mol Cell Endocrinol. 2012;356:24–30.CrossRefGoogle Scholar
  24. 24.
    Gorre N, Adhikari D, Lindkvist R, et al. mTORC1 signaling in oocytes is dispensable for the survival of primordial follicles and for female fertility. PLoS One. 2014;9:e110491.CrossRefGoogle Scholar
  25. 25.
    Li J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, et al. Activation of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A. 2010;107:10280–4.CrossRefGoogle Scholar
  26. 26.
    Adhikari D, Gorre N, Risal S, et al. The safe use of a PTEN inhibitor for the activation of dormant mouse primordial follicles and generation of fertilizable eggs. PLoS One. 2012;7:e39034.CrossRefGoogle Scholar
  27. 27.
    Fukuhara A, Matsuda M, Nishizawa M, Segawa K, Tanaka M, Kishimoto K, et al. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science. 2005;307:426–30.CrossRefGoogle Scholar
  28. 28.
    Ognjanovic S, Bao S, Yamamoto SY, Garibay-Tupas J, Samal B, Bryant-Greenwood GD. Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes. J Mol Endocrinol. 2001;26:107–17.CrossRefGoogle Scholar
  29. 29.
    Curat CA, Wegner V, Sengenes C, et al. Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. Diabetologia. 2006;49:744–7.CrossRefGoogle Scholar
  30. 30.
    Shen CJ, Tsai EM, Lee JN, Chen YL, Lee CH, Chan TF. The concentrations of visfatin in the follicular fluids of women undergoing controlled ovarian stimulation are correlated to the number of oocytes retrieved. Fertil Steril. 2010;93:1844–50.CrossRefGoogle Scholar
  31. 31.
    Xiao J, Xiao ZJ, Liu ZG, et al. Involvement of dimethylarginine dimethylaminohydrolase-2 in visfatin-enhanced angiogenic function of endothelial cells. Diabetes Metab Res. 2009;25:242–9.CrossRefGoogle Scholar
  32. 32.
    Bae YH, Bae MK, Kim SR, Lee JH, Wee HJ, Bae SK. Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis. Biochem Biophys Res Commun. 2009;379:206–11.CrossRefGoogle Scholar
  33. 33.
    Adya R, Tan BK, Punn A, Chen J, Randeva HS. Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signaling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res. 2008;78:356–65.CrossRefGoogle Scholar
  34. 34.
    Park JW, Kim WH, Shin SH, et al. Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway. Biochim Biophys Acta. 1813;2011:763–71.Google Scholar
  35. 35.
    Age Converter; mouse age calculator. Available from:
  36. 36.
    Castellano JM, Mosher KI, Abbey RJ, McBride A, James ML, Berdnik D, et al. Human umbilical cord plasma proteins revitalize hippocampal function in aged mice. Nature. 2017;544:488–92.CrossRefGoogle Scholar
  37. 37.
    Duncan FE, Gerton JL. Mammalian oogenesis and female reproductive aging. Aging. 2018;10:162–3.CrossRefGoogle Scholar
  38. 38.
    Shimazu T, Jiang JY, Liijima K, et al. Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature glits. Biol Reprod. 2003;69:1388–93.CrossRefGoogle Scholar
  39. 39.
    Danforth DR, Arbogast LK, Ghosh S, Dickerman A, Rofagha R, Friedman CI. Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary. Biol Reprod. 2003;68:1736–41.CrossRefGoogle Scholar
  40. 40.
    Iijima K, Jiang JY, Shimazu T, et al. Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats. J Reprod Dev. 2005;51:161–8.CrossRefGoogle Scholar

Copyright information

© Society for Reproductive Investigation 2020

Authors and Affiliations

  • Byung-Kyoo Park
    • 1
  • Min Jung Park
    • 2
  • Hwi Gon Kim
    • 1
  • Si Eun Han
    • 1
  • Chang-Woon Kim
    • 3
  • Bo Sun Joo
    • 2
    • 4
    Email author
  • Kyu-Sup Lee
    • 1
    Email author
  1. 1.Department of Obstetrics and GynecologyPusan National University School of MedicineBusanRepublic of Korea
  2. 2.The Korea Institute for Public Sperm BankBusanRepublic of Korea
  3. 3.Department of Obstetrics and Gynecology, Samsung Changwon HospitalSungkyunkwan University School of MedicineChangwonRepublic of Korea
  4. 4.Infertility InstitutePohang Women’s HospitalPohangRepublic of Korea

Personalised recommendations