1. Methods Objectives Results Conclusions References References
The Effect Of Allogeneic Mouse Serum, Fetal Bovine Serum And Bovine Serum Albumin On Sperm Capacitation
Omid Shahsavand Inanloo1,*, Homa Mohseni kouchesfahani1, Kazem Parivar1, Elaheh Amini2
1. Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
2. Department of Cell & Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
Objectives
In brief, 10 µl of MTT stock solution (5 mg/ml Embryocul) was added to each tube containing sperm suspension and incubated at 37oC in CO2 incubator for 1 hr. The tubes centrifuged at 6000 rpm for 6 min and the precipitate was dissolved in 200 µl dimethyl sulfoxide (DMSO). 100 µl of the purple solution were transferred into a 96-well plate and absorbance was measured using ELISA reader at 570nm wavelength.
Fertilization is a unique and amazing process involving two morphologically distinct cells, the sperm and the oocyte, which are recognized and fused together. This process begins when the sperm starts to penetrate the oocyte envelope and plasma membrane and ends in the exchange of maternal and paternal chromosomes, forming the zygote. The sperm must undergo functional changes following its genesis and subsequent maturation in the epididymis. Only sperm that have become capacitated can recognize and bind to the zona pellucida (ZP). The interaction between the sperm and the ZP initiates a signal transduction process resulting in exocytosis of the acrosomal contents during the acrosome reaction (AR) . To keep spermatozoa mobility for a long time and to evaluate the migration and fertilization, it is necessary to use a basic medium with nutrient factors. For this purpose, FBS is often used to maintain cell growth. The aim of this study is to find out more about the effect of allogeneic mouse serum (AMS) as compared with FBS and BSA on sperm capacitation.
Results
Conclusions
Results of MTT assay and motility were intermittent so that The MTT assay showed that sperm had the highest viability in 15% BSA. Also, the rate of progression in the medium containing 10% BSA and 5% FBS showed the highest percentage of progression and the highest percentage of motility was observed in medium containing 10% FBS and 5% AMS. In addition, there was a significant difference (p<0.05) between these groups and the control group.
Different types of serum are added to the medium as a supplement to enhance the quality of culture. The results indicate that albumin in the serum reduces cholesterol in the sperm membrane. The presence of serum and serum type are generally important for sperm capacitation in vitro. Moreover, low serum percentages show better results rather than other percentages in sperm capacitation.
References
Eskandari F, Momeni HR. Protective effect of silymarin on viability, motility and mitochondrial membrane potential of ram sperm treated with sodium arsenite. International Journal of Reproductive BioMedicine Jun;14(6):397.
Parodi J. Motility, viability, and calcium in the sperm cells. Systems biology in reproductive medicine Apr 1;60(2):65-71.
Nasr-Esfahani MH, Aboutorabi R, Esfandiari E, Mardani M. Sperm MTT viability assay: a new method for evaluation of human sperm viability. Journal of assisted reproduction and genetics Oct 1;19(10):
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods Dec 16;65(1-2):55-63.
World Health Organisation. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge university press; 1999 May 13.
Methods
epididymis of mice was extracted and transferred to Embryocul medium with 0, 5, 10, 15, 20 percentages of AMS, FBS and BSA. After treatment, Evaluation of sperm motility was done according to WHO guidelines. In brief, 10 µl of sperm suspension was placed on a counting-chamber. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), nonprogressive motile sperm (NPMS) and nonmotile sperm (NMS). This assay was performed based on a method described by Mosmann.
References