Uzun süreli inkübasyonun ve sıcaklığın oosit aktivatörü fosfolipaz C-zeta aktivitesine etkisi

Abstract

Amaç: Kapasite olmuş spermin fertilizasyon kapasitesinin

zamanla üstsel olarak düştüğü bilinmesine rağmen bunun nedeni

hala araştırılmaktadır. Çalışmada amaç; uzamış inkübasyonun

ve sıcaklığın sperm fertilizasyon kapasitesi üzerinde etkisini

hareketlilik ve oosit aktivasyon faktörü olarak kabul edilen

fosfolipaz C-zeta (PLCZ) ile değerlendirmektir.

Gereç ve Yöntem: On altı infertil erkek hastadan, ciddi

oligoasthenospermisi olmayan 11’inin semenlerine dansite

gradyan yıkaması uygulandı. Ardından örnekler bölünerek 3 gün

oda sıcaklığında veya 37°C’de kültüre edildi. Örnekler hareketlilik,

PLCZ boyanma yüzdesi ve yoğunluğu açısından günlük olarak

değerlendirildi.

Bulgular: Hem oda sıcaklığı, hem de vücut sıcaklığında her

üç parametrede de zamanla azalma gözlendi. Özellikle günler arası

hareketlilikteki değişim ile PLCZ boyanan sperm yüzdesindeki

değişim arasında güçlü bir korelasyon bulundu, ancak bu

korelasyon zamanla azalmakta idi.

Sonuç: İnkübasyon süresinin uzamasıyla, PLCZ boyanma

parametreleri ve hareketlilik arasında korelasyon görülmektedir.

PLCZ boyanmasının semen analizi ile rutinde kullanımı, özellikle

açıklanamayan infertilite ve fertilizasyon başarısızlığı vakalarının

spermlerinin fertilizasyon kapasitesinin belirlenmesinde yarar

sağlayabilecek ve yardımcı üreme teknileri (YÜT) sikluslarındaki

başarıyı artırabilecektir.

Keywords

• Sıcaklık,Fosfolipaz C-zeta,Fertilizasyon kapasitesi,Sperm,Oosit aktivasyonu,Uzun süreli inkübasyon

The effect of prolonged incubation and temperature on oocyte activator phospholipase C-zeta activity of sperm

Abstract

Objective: Fertilization capacity of capacitated sperm decreases

exponentially over time, but the reason is still under investigation.

The aim of the study was to analyze the effect of prolonged

incubation and temperature on sperm fertilization capacity with

motility and staining parameters of phospholipase C-zeta (PLCZ),

which is considered to be the oocyte activation factor.

Materials and Methods: Density gradient washing

was applied to semen of 11 infertile patients without severe

oligoasthenospermia out of 16 patients. The samples were divided

and cultured either at room temperature or at 37°C for 3 days. The

spermatozoons were evaluated for motility, PLCZ staining and

intensity daily.

Results: All parameters decreased both at room and body

temperature with increased incubation time. There was a strong

correlation between the change in motility and in the percentage

of PLCZ stained sperms, but this correlation decreased with

incubation time.

Conclusion: Prolonged incubation results show the correlation

between PLCZ staining parameters and motility. Routine use of

PLCZ staining together with semen analysis, will be useful to

predict fertilization capacity of the sperm especially for unexplained

infertility and fertilization failure cases, and also can increase the

success of assisted reproductive technologies (ART) cycles.

Keywords

• Temperature,Phospholipase C zeta,Fertilization capacity,Sperm,Oocyte activaton,Prolonged incubation

References

1. Weinberg CR, Wilcox AJ. A model for estimating the potency and survival of human gametes in vivo. Biometrics 1995;51:405-12. doi: 10.2307/2532929
2. Petrella C, Hsieh J, Blake E, Thrift K, Zacur H, Zhao Y. Human sperm can survive at room temperature for weeks: Measured by motility and viability of sperm maintained under various conditions. Fertil Steril 2003;80:S210. doi: 10.1016/S0015-0282(03)01468-7
3. Carlsen E, Giwercman A, Keiding N, Skakkebæk NE. Evidence for decreasing quality of semen during past 50 years. BMJ 1992;305(6854):609-13. doi: 10.1136/ bmj.305.6854.609
4. Organization WHO. WHO Laboratory Manual for the Examination and Processing of Human Semen. 2010.
5. Ribbes H, Plantavid M, Bennet PJ, Chap H, Douste- Blazy L. Phosopholipase C from human sperm specific for phsophoinositides. Biochim Biophys Acta 1987;919:245-54. doi: 10.1016/0005-2760(87)90264-5
6. Saunders CM, Larman MG, Parrington J, et al. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 2002;129:3533-44. doi: 10.3410/f.1008656.109808
7. Heytens E, Parrington J, Coward K, et al. Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod 2009;24:2417- 28. doi: 10.1093/humrep/dep207
8. Yelumalai S, Yeste M, Jones C, et al. Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection. Fertil Steril 2015;104:561- 8. doi: 10.1016/j.fertnstert.2015.05.018

9. Sobrero AJ, MacLeod J. The immediate postcoital test. Fertil Steril 1962;13:184-9. doi: 10.1016/S0015-0282(16)34447-8
10. Rubenstein BB, Strauss H, Lazarus ML, Hankin H. Sperm survival in women: Motile sperm in the fundus and tubes of surgical cases. Fertil Steril 1951;2:15-9. doi: 10.1016/S0015- 0282(16)30421-6
11. Ahlgren M. Sperm transport to and survival in the human fallopian tube. Gynecologic and Obstetric Investigation. 1975;6(3-4):206-14. doi: 10.1159/000301517
12. Settlage DSF, Motoshima M, Tredway DR. Sperm transport from the external cervical os to the fallopian tubes in women: a time and quantitation study. Fertil Steril 1973;24:655-61. doi: 10.1016/S0015-0282(16)39908-3
13. Barratt C, Cooke I. Review sperm transport in the human female reproductive tract—a dynamic interaction. Int J Androl 1991;14:394-411. doi: 10.1111/j.1365-2605.1991. tb01268.x
14. Perloff WH, Steinberger E. In vivo survival of spermatozoa in cervical mucus. Am J Obstet Gynecol 1964;88:439-42. doi: 10.1016/0002-9378(64)90499-5
15. Donnelly ET, Lewis SE, McNally JA, Thompson W. In vitro fertilization and pregnancy rates the influence of sperm motility and morphology on IVF outcome. Fertil Steril 1998;70:305-14. doi: 10.1016/S0015-0282(98)00146-0
16. Thijssen A, Klerkx E, Huyser C, Bosmans E, Campo R, Ombelet W. Influence of temperature and sperm preparation on the quality of spermatozoa. Reprod Biomed Online 2014;28:436-42. doi: 10.1016/j.rbmo.2013.12.005
17. Cohen J. Interaction between zona-free hamster eggs and human spermatozoa: Thesis, Erasmus University, The Netherlands: 1982.
18. Kashir J, Heynen A, Jones C, et al. Effects of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa. Reprod Biomed Online 2011;23:263-7. doi: 10.1016/j.rbmo.2011.04.006
19. Ferrer-Vaquer A, Barragan M, Freour T, Vernaeve V, Vassena R. PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI. J Assist Reprod Genet 2016;33:747-56. doi: 10.1007/s10815-016-0718-0
20. Appell RA, Evans PR. The effect of temperature on sperm motility and viability. Fertil Steril 1977;28:1329-32. doi: 10.1016/S0015-0282(16)42978-X
21. Kashir J, Jones C, Mounce G, et al. Variance in total levels of phospholipase C zeta (PLC-zeta) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability. Fertil Steril 2013;99:107-17. doi: 10.1016/j.fertnstert.2012.09.001