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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 31  |  Issue : 3  |  Page : 158-161

Comparison of the risk of smoking on proprotein convertase subtilisin/kexin level in the plasma membrane of human sperm


1 Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh Province, Indonesia
2 Biochemistry Laboratory, Chemistry Department, Universitas Brawijaya, Malang, Indonesia

Date of Web Publication17-Aug-2017

Correspondence Address:
D Dahril
Jalan Teuku Nyak Arief, Banda Aceh 23111, Aceh Province
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jms.jms_57_16

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  Abstract 


Context: Smoking is an abhorrent habit mostly performed by some people in developing countries like Indonesia. Cigarette smoke is believed to cause respiratory disorders and diseases as well as problems of reproduction and infertility, especially for men. Previous research has studied about proprotein convertase subtilisin/kexin type 4 (PCSK4) that plays a role in the reproduction system.
Aims: The present study is aimed at investigating the correlation between cigarette and disorder in PCSK4 molecule in the human sperm during fertilization process.
Settings and Design: The study was conducted in Indonesia, one of developing countries where the number of smokers keeps increasing. The prospective study employs cross-sectional approach to compare the PCSK4 level in the spermatozoa of male smokers and nonsmokers.
Materials and Methods: Spermatozoa ejaculation from ten individuals from both groups at productive age is collected. The enzyme-linked immunosorbent assay (ELISA) is carried out to compare the PCSK4 activity in both sample data.
Statistical Analysis Used: The Shapiro–Wilk Method is employed to analyze the data distribution. Moreover, t-test is also used to investigate equality of means.
Results: The finding indicates that based on ELISA, the PCSK4 level of male smokers is lower than that of nonsmokers.
Conclusions: It is concluded that smoking can decrease the PCSK4 activities in sperm which eventually influence the fertilization process.

Keywords: Cigarette smoke, proprotein convertase subtilisin/kexin type 4, reproduction


How to cite this article:
Dahril D, Aulanni'am A, Mutiawati V K. Comparison of the risk of smoking on proprotein convertase subtilisin/kexin level in the plasma membrane of human sperm. J Med Soc 2017;31:158-61

How to cite this URL:
Dahril D, Aulanni'am A, Mutiawati V K. Comparison of the risk of smoking on proprotein convertase subtilisin/kexin level in the plasma membrane of human sperm. J Med Soc [serial online] 2017 [cited 2017 Oct 17];31:158-61. Available from: http://www.jmedsoc.org/text.asp?2017/31/3/158/211101




  Introduction Top


Smoking is a common health issue in Indonesia and throughout the world because cigarette is one of the main causes for some chronic diseases such as infertility in men.[1],[2] Number of active smokers in developed countries is decreasing; however, it tends to increase in developing countries. The World Health Organization (WHO) states that level of intelligence and knowledge that are related to smoking habit in the society become the foundation for such high and dangerous smoking habit. The 1995 Household Health Survey and National Socioeconomic Survey showed that active smokers began to smoke at young age that is between 20 and 25 years and their smoking habit gradually increases until they are between 40 and 44 years.[2]

Infertility is a global phenomenon; it is predicted that 10% people at productive age have infertility. WHO data claim there are 60–80 million couples in the world who have to deal with infertility. Infertility has issue for 15% couples where male plays an important role.[3],[4] Defect in the function of sperm is the most frequent cause of infertility in men and the condition is difficult to take care of. Some factors that lead to defect in the function of sperm are physiological factor, genetic factor, oxidative stress, environment, and lifestyle. Infertility begins with decrease in the function of sperm that is indicated by decreasing quality of sperm.[5]

Decreasing quality of sperm is the main cause of infertility in 30% couples. Some studies revealed that cigarette may affect sperm quality, for example, asthenozoospermia, teratozoospermia, and oligozoospermia that occur in heavy smokers or long-term smokers. Exposure to the chemical substance in the cigarette may result in reactive oxygen species (ROS) which is a disorder of sperm that contains. Increasing ROS may cause damage in sperm deoxyribonucleic acid (DNA).[6]

Proprotein convertase subtilisin/kexin type 4 (PCSK4) molecule is the result of human spermatozoa isolation; the size of the molecule is 54 kDa expressed by plasma membrane of sperm acrosome.[7] PCSK4 molecule is found in testicular germinal cells and sperm is an important enzyme in reproduction process.[8] The molecule has pivotal role in the process of acrosome reaction through endoproteolytic that changes protein precurore into protein bioactive as the substance needed in penetration of pellucida ovary zone.[9] Studies conducted by Seidah and Prat[10] state that PCSK4 is known as one of the nine families of calcium-dependent serine endoproteinases that have role in proteolytic activation from secretory precursor protein into bioactive protein.[8] Changes in expression or activity of PCSK4 is predicted to be the cause of infertility issue for some infertility cases in human; however the mechanism has yet been predicted until now.[8] Till now, there has yet been a study about the correlation between cigarette and disorder in a PCSK4 molecule in human sperm during fertilization process.


  Materials and Methods Top


The study is a prospective preliminary study with cross-sectional approach and was conducted in 8 months from October 2013 to June 2014. There were two groups of samples, smokers and nonsmokers. The group consisting of nonsmokers was determined as the control group. The samples were spermatozoa ejaculation from ten individuals, ranging from 25 to 40 years, in each group. The criteria of inclusion of the samples possessed a healthy body and a result of individual semen analysis according to the criteria of WHO which had the semen volume of ± 1.5–2 ml per ejaculation, pH 7.2, spermatozoa concentration up to 15 × 106 spermatozoa per ml, a total number of 40 × 106 spermatozoa per ejaculation, motility up to 40%, normal morphology of 15%–30%, and vitality >50%.

Moreover, the individuals had to agree to give their semen for analysis after the informed consent was given. It was clearly declared in the consent that the researchers were to respect and appreciate the individuals in terms of not generating any pain on the subjects and distributing the result of the semen analysis to each involved individual. The consent also declared that the participation of the subjects was free of any charges. The informed consent was directly explained to the individuals before the research procedure began. This research was also approved by the Health Research Ethics Committee, Faculty of Medicine Padjadjaran University (No. 458/UN6.C2.1.2/KEPK/PN/2013).

Meanwhile, the criteria of exclusion of the samples were (1) not able to produce semen as demanded the analysis, (2) having medical record which is suspected to significantly influence the quality of the semen, and (3) withdrawing himself from participating in the research.

As the research procedure started, the semen that consisted of healthy sperm was collected from each of the subjects. The following step was carrying out NanoDrop Spectrophotometry examination to find out protein level on the tip of the spermatozoa.

It has been confirmed that spermatozoa as the result of ejaculation contains PCSK4 enzyme in acrosin spermatozoa based on immunocytochemistry method. In addition to measure level of PCSK4 in the samples of human spermatozoa, sandwich enzyme-linked immunosorbent assay (ELISA) was conducted using human PCSK4 (ELISA kit Cat. No. CSB-EL017643HU). Sandwich ELISA began with PCSK4 antigen coating. Standardized PCSK4 antigen as the result of isolation was taken for about 100 μL and then inserted into the well which has been labeled by antibody. The following step is incubation for 2 h at 37°C. The antigen that did link in the reaction was eliminated by washing. As many as 100 μL antibody-biotin was added into each well. The next step was 1-h incubation at 37°C. Re-washing using wash buffer was conducted to liquid as the result of the reaction from the well for three times. Having done that, 100 μL horseradish peroxidase-avidin was added to each well followed by 1-h incubation at 37°C. The samples were again rewashed using wash buffer for five times. Ninety microliters tetramethylbenzidine substrate was added to the well, followed by 15–30 min incubation at 37°C. The samples were not exposed to any light. Reaction in the sample was ended by adding 50 μL stop solution to each of the well. After 5 min, the reaction was analyzed using ELISA reader with 450 nm wavelength.

Meanwhile, the data analysis employed the one-way analysis of variance. When there was a significant difference, the analysis proceeded with Duncan test in the significant level of 5% with the help of SPSS 13.0 for Windows (IBM).


  Results and Discussion Top


Based on PCSK4 enzyme in the samples of human spermatozoa [Figure 1], it has been confirmed that all of the samples expressed PSCK4. PCSK4 is an enzyme that has been proven to be found in acrosome membrane of spermatozoa. The findings of the study reveal that it is found in the acrosome of the spermatozoa using immunocytochemical method [Figure 1]. The immunocytochemical test in the study used PSCK 4 monoclonal antibody used as ozospermatozoa antibody.
Figure 1: Proprotein convertase subtilisin/kexin type 4 expression on the surface of plasma membrane of spermatozoa (note: The arrows indicate proprotein convertase subtilisin/kexin type 4 expression on spermatozoa membrane)

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There is decreasing concentration of PCSK4 enzyme for the smokers [Table 1]. PCSK4 enzyme plays an important role in fertilization process that is as ligand from zona pellucida 3 (ZP3) molecules located in oocyte.
Table 1: Human PCSK4 Concentration of Non-Smoking and Smoking Groups

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The result of data distribution analysis using the Shapiro–Wilk method [Table 2] shows that the data are normally distributed. Smoking habit or cigarette has an influence in decreasing the concentration of PCSK4 in human spermatozoa. The analysis using t-test [Table 3] shows significant discrepancy, with P = 0.037 (P < 0.05). The data show that substances that cigarette smoke contains have been proven to affect the quality of spermatozoa based on the role of PCSK4 enzyme. PCSK4 enzyme is the one related to the motility of spermatozoa.
Table 2: Result of Saphiro-Wilk Normality Test

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Table 3: Result of t-Test

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Particles of cigarette smoke that damage one's health are nicotine, tar, and carbon monoxide. Cigarette smoke enters human body through inhalation and is filtered by the lungs. Numerous studies have published the effect of cigarette smoke toward one's health, two of which are respiratory and cardiovascular diseases. Cigarette smoke also has some effects toward male reproduction system, that is, a decrease in the sperm quality as the decreasing quality of sperm leads to a greater chance for fertilization.

A number of studies have shown that cigarette smoke may damage the DNA of spermatozoa; chromosome disorder is also shown during Golgi or spermatid phases through increase of stress oxidase. Such damage results in significant decrease in fertilization.[1],[6] Some factors that cause infertility in men damaged DNA due to cigarette smoke, oxidative substance, genetics, hormones, certain disease (such as diabetes and thyroid), and environmental and immunological disorder.[11],[12]

Based on the data analysis, it is revealed that PCSK4 level in smokers' spermatozoa is decreasing significantly. The theories have stated that PCSK4 is an enzyme that plays role in acrosome reaction through an endoproteolytic precursor protein process into bioactive protein, such as pro-pituitary adenylate cyclase activating polypeptide (PACAP) into PACAP active protein.[12] It is in line with the findings of El Mulla KF et al.'s study,[13] which concludes that acrosome reaction is significantly decreased in the spermatozoa samples of smokers.

Fertilization begins with sperm capitation process in the female tractus genitalia; it requires 3–24 h span before achieving oocytes.[14] Capacitation change is biochemistry and biophysic process on the surface of spermatozoa membrane that involves glycoprotein, decapacitation factor, acrosome stabilizing factor, and acrosome inhibitor.[14] Ties between membrane plasma of the spermatozoa and pellucide zone are the beginning of acrosome reaction that leads to intracellular signals that lead to acrosomal exocytosis, as seen in [Figure 2].[8],[15],[16]
Figure 2: Mammal fertilization

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  Conclusions Top


Based on the findings, it is proven that cigarette smoking decreases PCSK4 enzyme activities in the spermatozoa. It is ZP3 molecule ligand from the egg so that it interferes the fertilization process.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Swan SH, Elkin EP, Fenster L. The question of declining sperm density revisited: An analysis of 101 studies published 1934-1996. Environ Health Perspect 2000;108:961-6.  Back to cited text no. 12
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El Mulla KF, Kohn FM, El Beheiry AH, Schill WB. The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction. Hum Reprod 1995;10:3190-4.  Back to cited text no. 13
    
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Avella MA, Dean J. Fertilization with acrosome-reacted mouse sperm: Implications for the site of exocytosis. Proc Natl Acad Sci U S A 2011;108:19843-4.  Back to cited text no. 15
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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