Sains Malaysiana 47(6)(2018): 1101–1108

http://dx.doi.org/10.17576/jsm-2018-4706-04

Quantification of HSP70 Gene Expression and Determination of Capacitation Status of Magnetically Separated Cryopreserved Bovine Spermatozoa at Different Thawing

Temperature and Time

(Kuantifikasi Ekspresi Gen HSP70 dan Penentuan Status Kapasitasi Sperma Lembu

yang telah Dikrioawet pada Suhu Pencairan dan Masa yang Berbeza)

SYARIFAH FAEZAH SYED MOHAMAD^1*, SITI FATIMAH IBRAHIM³, NUR HILWAN ISMAIL², KHAIRUL OSMAN⁴, FARAH HANAN FATHIHAH JAAFAR³, CHEW FANG NANG³ & FARIDA ZURAINA MOHD YUSOF²

¹Department of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA

26400 Bandar Tun Abd Razak Jengka, Pahang Darul Makmur, Malaysia

²Department of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA

40450 Shah Alam, Selangor Darul Ehsan, Malaysia

³Department of Physiology, Faculty of Medicine, UKM Medical Centre, Level 18, Pre-Clinical Building

Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

⁴Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz

50300 Kuala Lumpur, Federal Territory, Malaysia

Received: 23 November 2015/Accepted: 10 January 2018

ABSTRACT

The role of heat shock protein in reproduction is widely known as a molecular chaperone in aiding and repairing protein formation when stress occurred. The present objectives were to evaluate the effect of different thawing temperature and time on the expression of HSP70 gene expression and the capacitation status in cryopreserved bovine spermatozoa. Briefly, fresh ejaculates were obtained from three different adult bulls. The semen then underwent a sperm washing technique known as Magnetic Activated Cell Sorting System (MACS) and later on, cryopreserved. The sperm- containing straws were then thawed at five different thawing temperatures and time post-cryostorage; 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s and 80°C for 5 s. The RNA was extracted from each of the sperm’s pellets and converted to cDNA prior to the qPCR process. Capacitation status was then determined by means of CTC assay. The results showed that after the process of amplification, there is a significant different of HSP70 gene expression in MACS process samples when the thawing process was performed at 37°C for 30 s, with p<0.05. Furthermore, the CTC assay also showed that thawing at the same temperature gave less capacitated spermatozoa with p<0.05. As a conclusion, MACS yield spermatozoa with a better expression of HSP70 gene and less capacitated spermatozoa when thawing was done at 37°C for 30 s.

Keywords: CTC; heat-shock protein; MAC; sperm cryopreservation; thawing

ABSTRAK

Peranan protein kejutan haba dalam pembiakan telah diketahui secara meluas terutamanya sebagai molekul pengiring dalam membantu dan memperbaik pembentukan protein apabila tekanan berlaku. Objektif semasa adalah untuk menilai kesan suhu dan masa pencairan yang berbeza pada ekspresi gen protein HSP70 dan status kapasitasi sperma lembu yang telah dikrioawet. Secara ringkas, ejakulasi segar diperoleh daripada tiga lembu jantan dewasa yang berbeza. Air mani kemudiannya menjalani teknik penyediaan sperma yang dikenali sebagai Magnetic Activated Cell Sorting System (MACS) dan kemudiannya dikrioawet. Selepas itu, straw yang mengandungi sperma kemudiannya dicairkan pada lima suhu pencairan dan masa yang berbeza; 20°C selama 13 s, 37°C selama 30 s, 40°C selama 7 s, 60°C selama 6 s dan 80°C selama 5 s. RNA kemudian diekstrak daripada pelet sperma dan ditukarkan kepada cDNA sebelum proses qPCR. Status kapasitasi kemudiannya telah dilakukan melalui esei CTC. Keputusan kajian menunjukkan bahawa selepas proses penguatan, ekspresi gen HSP70 terdapat perbezaan yang signifikan terhadap sperma yang telah menjalani MACS apabila pencairan dilakukan pada suhu 37°C selama 30 s dengan nilai p<0.05. Tambahan pula, asai CTC juga menunjukkan bahawa pencairan pada suhu yang sama memberikan sperma yang kurang berkapasiti dengan nilai p<0.05. Kesimpulannya, MACS boleh memberikan spermatozoa yang berkualiti baik dengan ekspresi HSP70 yang tinggi dan sperma yang kurang berkapasiti apabila pencairan dilakukan pada 37°C selama 30 s.

Kata kunci: CTC; krioawet sperma; MACS; pencairan; protein kejutan haba

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*Corresponding author; email: sharifahfaezah@pahang.uitm. edu.my