Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지

Effect of Cholesterol Supplementation in Freezing Medium on the Survival and Integrity of Human Sperm after Cryopreservation

콜레스테롤이 동결-해동 후 인간정자의 생존과 기능보존에 미치는 영향

  • Lim, Jung-Jin (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Sung, Su-Ye (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Kim, Kye-Seong (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University) ;
  • Song, Seung-Hon (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Lee, Woo-Sik (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Yoon, Tae-Ki (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Lee, Dong-Ryul (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University)
  • 임정진 (강남 차병원 여성의학연구소 불임의학연구실) ;
  • 성수예 (강남 차병원 여성의학연구소 불임의학연구실) ;
  • 김계성 (한양대학교 해부.세포 생물학교실) ;
  • 송승훈 (강남 차병원 여성의학연구소 불임의학연구실) ;
  • 이우식 (강남 차병원 여성의학연구소 불임의학연구실) ;
  • 윤태기 (강남 차병원 여성의학연구소 불임의학연구실) ;
  • 이동률 (강남 차병원 여성의학연구소 불임의학연구실)
  • Published : 2008.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Objective: During cryopreservation process, cold shock and cryo-injury affect the fertilizing capacity of the sperm by damaging cell membranes with loss of functional integrity. A longstanding concept for preventing the cryo-damage is to stabilize the plasma membrane by incorporating cholesterol. This study was to determine the effects of cholesterol in freezing media on the motility and functional integrity of human sperm after cryopreservation. Methods: Control group (non-cholesterol treated) and different concentrations of cholesterol-treated sperm (14 healthy males) were frozen and thawed. After freezing and thawing of sperm, the quality of sperm was evaluated by sperm analysis, acrosome reaction test and sperm chromatin structure assay. Results: When human sperm were incubated in sperm freezing medium (SFM) containing $0.5{\mu}g$ cholesterol and then freezing/thawing, the motility of sperm have significantly improved compared to those untreated cholesterol ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). The rate of calcium ionophore-induced acrosome reactions in post-thawed sperm was significantly higher than that ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05) in SFM containing cholesterol. Sperm chromatin structure assay revealed that DNA damage to the sperm in the cholesterol-treated group was lower than that of non-treated group. Conclusion: These results suggest that increased cholesterol content of sperm plasma membrane by supplementation of cholesterol in SFM improves sperm motility, capacitation status, and DNA integrity. Therefore, addition of cholesterol into SFM could be a useful for protecting human sperm from cold shock and cryo-injury during cryopreservation.

목 적: 정자의 동결 과정에서 생길 수 있는 급격한 온도 차에 의한 동결 충격이나 동결 상해등에 의한 세포막의 손상, 세포의 기능 장애 등은 정자의 수정능에 영향을 미칠 수 있다. 본 연구에서는 인간 정자를 동결 보존하는 과정에서 콜레스테롤 전처리가 정자의 운동성 및 기능보존에 미치는 영향을 알아보고자 하였다. 연구방법: 본원을 내원한 14명 남성의 정자를 대상으로 콜레스테롤을 첨가하지 않은 대조군 (control)과 여러 농도의 콜레스테롤을 동결보존액에 첨가한 실험군에서 정자의 동결-융해 후 상태를 다음 3가지 방법으로 비교, 분석하였다. 1) 정자 분석, 2) calcium ionophore로 유도된 첨체 반응 검사, 3) 정자 염색질 구조 분석 (sperm chromatin structure assay). 결 과: 첫째로 인간 정자의 운동성은 $0.5{\mu}g$ 농도의 콜레스테롤을 첨가한 동결보존액에서 동결-해동하였을 경우, 콜레스테롤을 첨가하지 않은 군에 비해 유의적 차이를 보이며 증가하는 것을 확인하였다 ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). 다음으로 동된 정자의 첨체 반응 검사에서도 콜레스테롤을 첨가한 동결보존액에서의 첨체 반응이 일어나는 정자의 비율이 첨가하지 않은 군에 비해 유의하게 높게 관찰되었다 ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05). 마지막으로 정자 염색질 구조 분석에서는 콜레스테롤을 첨가한 군이 첨가하지 않은 군에 비해 정자의 DNA손상이 적게 나타남을 확인하였다. 결 론: 본 실험은 동결보존액을 통한 정자 원형질막 내 콜레스테롤 함유량의 증가가 동결-융해 후 정자의 운동성과 수정능(capacitation status)을 증가시키고 DNA 손상을 방지하는 역할을 한다는 결과를 보여주었다. 이러한 결과를 통해 동결보존액 내 콜레스테롤의 첨가는 인간 정자의 동결보존 동안 발생할 수 있는 동결 상해를 줄여줄 수 있는 유용한 방법으로 사료된다.

Keywords

References

  1. Morshedi M, Gosden RG. Cryopreservation of sperm. In: Tulandi T, Gosden RG, editor. Preservation of fertility. London: Taylor and Francis; 2004. p.111-24
  2. Moore AI, Squires EL, Graham JK. Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology 2005; 51: 241-9 https://doi.org/10.1016/j.cryobiol.2005.07.004
  3. Darin-Bennett A, Poulos A, White IG. The effect of cold shock and freeze-thawing on release of phospholipids by ram, bull, and boar sperm. Aust J Biol Sci 1973; 26: 1409-20 https://doi.org/10.1071/BI9731409
  4. Parks JE, Lynch DV. Lipid composition and thermotropic phase behavior of boar, bull, stallion and rooster sperm membranes. Cryobiology 1992; 29: 255-66 https://doi.org/10.1016/0011-2240(92)90024-V
  5. Mouritsen OG, Zuckermann MJ. What's so special about cholesterol? Lipid 2004; 39: 1101-13 https://doi.org/10.1007/s11745-004-1336-x
  6. Combes GB, Varner DD, Schroeder F, Burghardt RC, Blanchard TL. Effect of cholesterol on the motility and plasma membrane integrity of frozen equine sperm after thawing. J Reprod Fertil Suppl 2000; 56: 127-32
  7. Purdy PH, Graham JK. Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology 2004a; 48: 36-45 https://doi.org/10.1016/j.cryobiol.2003.12.001
  8. Purdy PH, Graham JK. Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility. Biol Reprod 2004b; 71: 522-7 https://doi.org/10.1095/biolreprod.103.025577
  9. Meseguer M, Garrido N, Martinez-Conejero MA, Simon C, Pellicer A, Remohi J. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004; 81: 588-94 https://doi.org/10.1016/j.fertnstert.2003.09.035
  10. Garner DL, Thomas CA, Joerg HW, DeJarnette JM, Marshall CE. Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine sperm. Biol Reprod 1997; 57: 1401-6 https://doi.org/10.1095/biolreprod57.6.1401
  11. Lee DR, Lee JE, Yoon HS, Roh SI. Induction of acrosome reaction in human sperm accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection. Fertil Steril 1997; 67: 315-20 https://doi.org/10.1016/S0015-0282(97)81917-6
  12. Evenson DP, Higgins PJ, Grueneberg D, Ballachey BE. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 1985; 6: 238-53 https://doi.org/10.1002/cyto.990060311
  13. Evenson DP, Jost LK, Baer RK, Turner TW, Schrader SM. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod Toxicol 1991; 5: 115-25 https://doi.org/10.1016/0890-6238(91)90039-I
  14. Visconti PE, Ning XP, Fornes MW, Alvarez JG, Stein P, Connors SA, et al. Cholesterol efflux mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. Dev Biol 1999; 214: 429-43 https://doi.org/10.1006/dbio.1999.9428
  15. Talbot P, Summers RG, Hylander BL, Keough EM, Franklin LE. The role of calcium in the acrosome reaction: an analysis using ionophore A23187. J Exp Zool 1976; 198: 383-92 https://doi.org/10.1002/jez.1401980312
  16. Nolan JP, Graham JK, Hammerstedt RH. Artificial induction of exocytosis in bull sperm. Arch Biochem Biophys 1992; 292: 311-22 https://doi.org/10.1016/0003-9861(92)90084-A
  17. Kramer RY, Garner DL, Bruns ES, Ericsson SA, Prins GS. Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human sperm. J Androl 1993; 14: 374-84
  18. Meistrich ML. Nuclear morphogenesis during spermiogenesis. In: de Kretser D, editor. Molecular Biology of the Male Reproductive System. San Diego: Academic Press; 1993. p.67-97
  19. Purdy PH, Fox MH, Graham JK. The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition. Cryobiology 2005; 51: 102-12 https://doi.org/10.1016/j.cryobiol.2005.05.004
  20. Ladbrooke BD, Williams RM, Chapman D. Studies on lecithincholesterol- water interactions by differential scanning calorimetry and X-ray diffraction. Biochim Biophys Acta 1968; 150: 333-40 https://doi.org/10.1016/0005-2736(68)90132-6