Effects of Heyan Kuntai Capsule (和颜坤泰胶囊) on Follicular Development and Oocyte Cohesin Levels in Aged Mice

  • Bin Zhang
  • Nan Chu
  • Xue-min Qiu
  • Wei Tang
  • Hans-Jürgen Gober
  • Da-jin Li
  • Ling Wang
Original Article



To evaluate the effect of Heyan Kuntai Capsule (和颜坤泰胶囊, HYKT) on the ovarian function of aged mice and expressions of cohesion complexes in oocytes.


Twenty-five 9-month-old female C57BL/6J mice were randomly divided into 5 groups by block randomization method (n=5 per group), including the control group (saline), 17 β-estradiol group [E2, 100 μg/(kg·d)], and low-, medium-, and high-dose of HYKT groups [0.3, 0.9, 2.7 g/(kg·d), respectively]. All mice were treated by intragastric administration for 4 weeks. Hematoxylin and eosin staining and anti-VASA staining were used to detect the amounts of follicles. The apoptosis of follicles was measured by anti-gamma H2A histone family member X (γH2AX) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The density of cohesin subunits, REC8 meiotic recombination protein (REC8), structural maintenance of chromosome (SMC) 1 β and SMC3 in oocytes were evaluated by immunofluorescent staining.


After administration of E2 and high-dose of HYKT, the total number of follicles as well as the number of primordial and primary follicles were significantly increased (P<0.05). Anti-γ H2AX staining and TUNEL assay demonstrated that high-dose of HYKT and E2 partly suppressed the apoptosis of follicles (P<0.05). Furthermore, it showed an increased trend in the levels of REC8 and SMC1 β, after administration with E2 and HYKT, and no obvious change in the level of SMC3.


HYKT could enhance the number of follicles, suppress apoptosis of oocytes and have a trend to elevate the meiotic-specific cohesin subunits (REC8 and SMC1 β) in oocytes of aged mice, indicating a beneficial effect on the ovarian function in terms of the quantity and quality of follicles.


follicular development cohesin oocyte Heyan Kuntai Capsule Chinese medicine 


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  1. 1.
    Te Velde ER, Pearson PL. The variability of female reproductive ageing. Hum Reprod Update 2002;8:141–154.CrossRefPubMedGoogle Scholar
  2. 2.
    Group ECW. Fertility and ageing. Hum Reprod Update 2005;11:261–276.CrossRefGoogle Scholar
  3. 3.
    Ottolenghi C, Uda M, Hamatani T, Crisponi L, Garcia J-E, Ko M, et al. Aging of oocyte, ovary, and human reproduction. Annals New York Acad Sci 2004;1034:117–131.CrossRefGoogle Scholar
  4. 4.
    Broekmans FJ, Knauff EaH, Te Velde ER, Macklon NS, Fauser BC. Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metabol 2007;18:58–65.CrossRefGoogle Scholar
  5. 5.
    Broer SL, Broekmans FJM, Laven JSE, Fauser BCJM. Anti-Müllerian hormone: ovarian reserve testing and its potential clinical implications. Hum Reprod Update 2014;20:688–701.CrossRefPubMedGoogle Scholar
  6. 6.
    Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991;129:2799–2801.CrossRefPubMedGoogle Scholar
  7. 7.
    Jiang JY, Cheung CKM, Wang Y, Tsang BK. Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia. Front Biosci 2003;8:d222–d237.CrossRefPubMedGoogle Scholar
  8. 8.
    Craig J, Orisaka M, Wang H, Orisaka S, Thompson W, Zhu C, et al. Gonadotropin and intra-ovarian signals regulating follicle development and atresia: the delicate balance between life and death. Front Biosci 2007;12:3628–3639.CrossRefPubMedGoogle Scholar
  9. 9.
    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;14:131–142.CrossRefPubMedGoogle Scholar
  10. 10.
    Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342–1346.CrossRefPubMedGoogle Scholar
  11. 11.
    Tilly JL. Commuting the death sentence: how oocytes strive to survive. Nat Rev Mol Cell Biol 2001;2:838–848.CrossRefPubMedGoogle Scholar
  12. 12.
    Fujino Y, Ozaki K, Yamamasu S, Ito F, Matsuoka I, Hayashi E, et al. Ovary and ovulation: DNA fragmentation of oocytes in aged mice. Hum Reprod 1996;11:1480–1483.CrossRefPubMedGoogle Scholar
  13. 13.
    Tatone C, Carbone MC, Gallo R, Delle Monache S, Di Cola M, Alesse E, et al. Age-associated changes in mouse oocytes during postovulatory in vitro culture: possible role for meiotic kinases and survival factor Bcl2. Biol Reprod 2006;74:395–402.CrossRefPubMedGoogle Scholar
  14. 14.
    Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2001;2:280–291.CrossRefPubMedGoogle Scholar
  15. 15.
    Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet 2012;13:493–504.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Haering CH, Löwe J, Hochwagen A, Nasmyth K. Molecular architecture of SMC proteins and the yeast cohesin complex. Mol Cell 2002;9:773–788.CrossRefPubMedGoogle Scholar
  17. 17.
    Parisi S, Mckay MJ, Molnar M, Thompson MA, Van Der Spek PJ, et al. REC8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans. Mol Cell Biol 1999;19:3515–3528.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Watanabe Y, Nurse P. Cohesin REC8 is required for reductional chromosome segregation at meiosis. Nature 1999;400:461–464.CrossRefPubMedGoogle Scholar
  19. 19.
    Pezzi N, Prieto I, Kremer L, Pérez Jurado LA, Valero C, Del Mazo J, et al. STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion. FASEB J 2000;14:581–592.CrossRefPubMedGoogle Scholar
  20. 20.
    Prieto I, Suja JA, Pezzi N, Kremer L, Martinez-A C, Rufas JS, et al. Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I. Nat Cell Biol 2001;3:761–766.CrossRefPubMedGoogle Scholar
  21. 21.
    Revenkova E, Eijpe M, Heyting C, Gross B, Jessberger R. Novel meiosis-specific isoform of mammalian SMC1. Mol Cell Biol 2001;21:6984–6998.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chiang T, Schultz RM, Lampson MA. Meiotic origins of maternal age-related aneuploidy. Biol Reprod 2012;86:1–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Jessberger R. Age-related aneuploidy through cohesion exhaustion. EMBO Reports 2012;13:539–546.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Jones KT, Lane SIR. Chromosomal, metabolic, environmental, and hormonal origins of aneuploidy in mammalian oocytes. Exp Cell Res 2012;318:1394–1399.CrossRefPubMedGoogle Scholar
  25. 25.
    Herbert M, Kalleas D, Cooney D, Lamb M, Lister L. Meiosis and maternal aging: insights from aneuploid oocytes and trisomy births. Cold Spring Harbor Perspect Biol 2015;7:a017970.CrossRefGoogle Scholar
  26. 26.
    Yang YF. RP-HPLC determination of paeonifl orin, baicalin, palmatine hydrochloride and berberine hydrochloride in Kuntai Capsules. Chin Traditional Patent Med (Chin) 2010;32:958–960.Google Scholar
  27. 27.
    Li HF. Simultaneous determination of 6 components in Kuntai Capsule by RP-HPLC wavelength switching method. China Mod Med (Chin) 2015;22:7–11.Google Scholar
  28. 28.
    Zhang QY, Wang JW, Yu JM. Efficacy and safety of Kuntai Capsule and estrogen for menopausal syndrome: a metaanalysis. Natl Med J China (Chin) 2013;93:3445–3449.Google Scholar
  29. 29.
    Li CC, Wang JJ, Chen C, Li YF, Zheng QS, Yang J, et al. Treating menopause syndrome by Kuntai Capsule and hormone replacement therapy: a meta-analysis of efficacy and safety comparison. Chin J Integr Tradit West Med (Chin) 2013;33:1183–1190.Google Scholar
  30. 30.
    Zhang SF, Liu Y, Xie Q, Bi CP, Zhu HT, Ha LX, et al. Effects of Gengnian Ningxin Capsule on the histomorphology of ovarin and uterine in the menopausal rats. Chin J Geriatr (Chin) 2004;23:648–651.Google Scholar
  31. 31.
    Lian F, Jiang XY. Effect of Kuntai Capsule on the number of retrieved oocytes, high-quality oocytes and embryos in in vitro fertilization of poor ovarian response patients. Chin J Integr Tradit West Med (Chin) 2014;34:917–921.Google Scholar
  32. 32.
    Li WW, An LH, Zhang G, Wang XL, Sun LJ, Guan YC, et al. The Combined application of estradiol valerate-cyproterone acetate and Kuntai Capsule in poor ovarian response patients in vitro fertilization. J Pract Obestet Gynecol (Chin) 2014;30:681–685.Google Scholar
  33. 33.
    Chang EM, Han JE, Won HJ, Kim YS, Yoon TK, Lee WS. Effect of estrogen priming through luteal phase and stimulation phase in poor responders in in-vitro fertilization. J Assist Reprod Genet 2012;29:225–230.CrossRefPubMedGoogle Scholar
  34. 34.
    Wang CC, Li L, Tang LY, Leung PC. Safety evaluation of commonly used Chinese herbal medicines during pregnancy in mice. Hum Reprod 2012;27:2448–2456.CrossRefPubMedGoogle Scholar
  35. 35.
    You SL, ed. Experimental zoology of obstetrics and gynecology. Beijing: Chinese Medicine Publisher of China;2007:7–8.Google Scholar
  36. 36.
    Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 1996;17:121–55.CrossRefPubMedGoogle Scholar
  37. 37.
    Skaznik-Wikiel ME, Mcguire MM, Sukhwani M, Donohue J, Chu T, Krivak TC, et al. Granulocyte colony-stimulating factor with or without stem cell factor extends time to premature ovarian insufficiency in female mice treated with alkylating chemotherapy. Fertil Steril 2013;99:2045–2054.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhang JM, Li LX, Yang YX, Liu XL, Wan XP. Is caspase inhibition a valid therapeutic strategy in cryopreservation of ovarian tissue? J Assist Reprod Genet 2009;26:415–420.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Tsutsumi M, Fujiwara R, Nishizawa H, Ito M, Kogo H, Inagaki H, et al. Age-related decrease of meiotic cohesins in human oocytes. PLoS One 2014;9:e96710.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Baker TG. A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society of London. Series B, Biological Sciences 1963;158:417–433.CrossRefPubMedGoogle Scholar
  41. 41.
    Billig H, Chun S, Eisenhauer K, Hsueh A. Gonadal cell apoptosis: hormone-regulated cell demise. Hum Reprod Update 1996;2:103–117.CrossRefPubMedGoogle Scholar
  42. 42.
    Ansari RM. Potential use of durian fruit (Durio zibenthinus Linn) as an adjunct to treat infertility in polycystic ovarian syndrome. J Integr Med 2016;14:22–28.CrossRefPubMedGoogle Scholar
  43. 43.
    Koebele SV, Bimonte-Nelson HA. Modeling menopause: the utility of rodents in translational behavioral endocrinology research. Maturitas 2016;87:5–17.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Albamonte MS, Willis MA, Albamonte MI, Jensen F, Espinosa MB, Vitullo AD. The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis. Hum Reprod 2008;23:1895–1901.CrossRefPubMedGoogle Scholar
  45. 45.
    Dutta S, Sengupta P. Men and mice: relating their ages. Life Sci 2016;152:244–248.CrossRefPubMedGoogle Scholar
  46. 46.
    Roti Roti EC, Leisman SK, Abbott DH, Salih SM. Acute doxorubicin insult in the mouse ovary is cell- and follicle-type dependent. PLoS One 2012;7:e42293.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Zhang SF, Ha LX, Xie Q, Jiang J, Liu Y, Xia X, et al. Effect of Gengnian Ningxin Capsule and estradial valerate on splenocyte apoptosis gene Bcl-2 and Bax expression of menopausal rats. Chin J Integr Tradit West Med (Chin) 2004;24:201–204.Google Scholar
  48. 48.
    Cun-Cun L, Jing-Jing W, Chao C, Yun-Fei L, Qing-Shan Z, Juan Y, et al. Clinical efficacy of Kuntai Capsule compared with estrogen progesterone sequential treatment on perimenopausal syndrome and impact on life quality. Chin J Biochem Pharm (Chin) 2013;33:1183–1190.Google Scholar
  49. 49.
    Garcia-Cruz R, Brieno MA, Roig I, Grossmann M, Velilla E, Pujol A, et al. Dynamics of cohesin proteins REC8, STAG3, SMC1β and SMC3 are consistent with a role in sister chromatid cohesion during meiosis in human oocytes. Hum Reprod 2010;25:2316–2327.CrossRefPubMedGoogle Scholar
  50. 50.
    Chiang T, Duncan FE, Schindler K, Schultz RM, Lampson MA. Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes. Curr Biol 2010;20:1522–1528.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Lister LM, Kouznetsova A, Hyslop LA, Kalleas D, Pace SL, Barel JC, et al. Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2. Current Biol 2010;20:1511–1521.CrossRefGoogle Scholar
  52. 52.
    Chiang T, Schultz RM, Lampson MA. Age-dependent susceptibility of chromosome cohesion to premature separase activation in mouse oocytes. Biol Reprod 2011;85:1279–1283.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Merriman JA, Jennings PC, Mclaughlin EA, Jones KT. Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months. Biol Reprod 2012;86:49.CrossRefPubMedGoogle Scholar
  54. 54.
    Revenkova E, Eijpe M, Heyting C, Hodges CA, Hunt PA, Liebe B, et al. Cohesin SMC1 β is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination. Nat Cell Biol 2004;6:555–562.CrossRefPubMedGoogle Scholar
  55. 55.
    Hodges CA, Revenkova E, Jessberger R, Hassold TJ, Hunt PA. SMC1 β-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction. Nat Genet 2005;37:1351–1355.CrossRefPubMedGoogle Scholar
  56. 56.
    Koehler KE, Schrump SE, Cherry JP, Hassold TJ, Hunt PA. Near-human aneuploidy levels in female mice with homeologous chromosomes. Current Biol 2006;16:R579–R580.CrossRefGoogle Scholar
  57. 57.
    Selesniemi K, Lee HJ, Muhlhauser A, Tilly JL. Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies. Proc Natl Acad Sci U S A 2011;108:12319–12324.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Chinese Association of the Integration of Traditional and Western Medicine 2018

Authors and Affiliations

  • Bin Zhang
    • 1
    • 2
    • 3
  • Nan Chu
    • 1
    • 2
    • 3
  • Xue-min Qiu
    • 1
    • 2
    • 3
  • Wei Tang
    • 4
  • Hans-Jürgen Gober
    • 5
  • Da-jin Li
    • 1
    • 2
    • 3
  • Ling Wang
    • 1
    • 2
    • 3
  1. 1.Institute of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan UniversityShanghaiChina
  2. 2.The Academy of Integrative Medicine of Fudan UniversityShanghaiChina
  3. 3.Shanghai Key Laboratory of Female Reproductive Endocrine Related DiseasesShanghaiChina
  4. 4.Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicinethe University of TokyoTokyoJapan
  5. 5.Department of PharmacyKepler University Clinic, Neuromed CampusLinzAustria

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