Correlation between creatine kinase activity, lipid-peroxidation and water test in male infertility.

SP Dandekar, GM Parkar 
 Department of Biochemistry, Seth G.S. Medical College and K.E.M. Hospital, Parel, Mumbai, India., India

Correspondence Address:
S P Dandekar
Department of Biochemistry, Seth G.S. Medical College and K.E.M. Hospital, Parel, Mumbai, India.
India

Full Text

There is a constant need to identify cellular markers of sperm quality so that in addition to their diagnostic value, they may facilitate the identification of specific deficiencies of sperm-function[1]. Therefore the activity of creatine kinase (CK), the key enzyme in synthesis and transport of energy[2],[3],[4], was studied in correlation with sperm concentration, water test and lipid peroxidation.

Studies on the enzymatic status of spermatozoa are specialized and are limited to a few enzymes. The fertilizing capacity of a sperm sample is usually estimated by the concentration of spermatozoa and their motility and morphology. There are, however, some samples which while having normal spermiograms are infertile. It would thus appear that the classical, biological criteria of fertility are sometimes insufficient and might be overcome by some biochemical criteria. The enzymatic profile of spermatozoa should constitute a good indication of functional metabolic activity. The enzymes present in seminal fluid are shown to be derived from secretions of seminiferous tubules, spermatozoa, epididymis, seminal vesicles and prostate gland[5]. Thus, the estimation of different enzymes in semen permits one to obtain markers of seminal quality[6]. The neutral enzyme a - glucosidase, found in semen, is essentially secreted by the epididymis in particular by its- part and is shown to be a marker of occlusion at this level[7].

Creatine kinase (E.C.2.7.3.2) catalyses the reversible phosphorylation of their ADP to ATP or creatine to creatine phosphate, thus maintaining an immediately accessible energy reservoir in the cell[8]. Cells requiring high energy such as spermatozoa are characterized by high creatine kinase in human spermatozoa are characterised by high creatine kinase activity. It is generally accepted that there are two isoenzymes of creatine kinase in human spermatozoa, designated as CK-BB brain type) and CK-MM (muscle type). Although the presence of CK-BB in human spermatozoa is not disputed[9],[10], the presence of the second isoenzyme is still controversial[11].

In the study, the presence of isoform CK-MB was found in human sperm samples and was measured spectrophotometrically. The ATP is supplied by creatine phosphokinase, which is the key enzyme in transport and synthesis of energy[10]. [Figure:1]

Evidence for the hypothesis has come from a number of independent studies indicating that sperm function is frequently associated with elevated activities of certain key enzymes including creatine kinase[2],[12],[13]. These enzymes are not thought to be directly responsible for loss of sperm-function but rather to act as biochemical markers of normality of sperm differentiation. Errors of spermiogenesis can result in creation of oxidative stress. This could be proved by showing a positive association between the CK content of human spermatozoa and induction of peroxidative damage[11].

Lipid peroxidation may be defined as oxidative deterioration of polyunsaturated acids it is a physiological phenomenon occurring in all cells that are rich in lipid, specially the polyunsaturated fatty acids. Lipid peroxidation plays a significant role in the etiology of defective sperm-function[14]. The onset of lipid peroxidation in susceptible forms leads to the progressive accumulation of lipid hydroperoxides in the sperm plasma membrane, which decomposes to form malonaldehyde (MDA) which is an index of lipid peroxidative damage[15],[16].

The CK activity was also studied in correlation with the water test, to detect a marker for sperm-membrane integrity. The functional competence of human sperm-membrane is assessed by studying the swelling reactions of sperm when suspended in a medium of distilled water. Lomeo and Giambersio[17] developed a simpler technique, the water test to detect sperm-membrane integrity. Sperm-swelling generally indicates normal sperm membrane function and the transport of molecules across the sperm-membrane when exposed to hypo-osmotic swelling conditions. Membrane integrity is not only important for sperm metabolism but also a correct change in the properties of membrane is required for successful union of male and female gamete i.e. for sperm capacitation, the acrosome reaction and the binding of spermatozoon to egg surface. This study therefore attempts to correlate creatine kinase activity with lipid peroxidation and the water test.

::   Material and method

A total of 80 samples were collected and categorized into normal oligospermic and azoospermic samples. The age group chosen was between 26 years to 40 years. All the samples were collected from married patients attending the infertility clinics. Ejaculates were obtained by masturbation after three days of sexual abstinence. Samples were collected in sterile wide mouthed containers and after liquefaction. Analysis was performed according to WHO methods and standards[18]. The ejaculates of the normospermic group had a sperm count of >30 million per ml, and >50 % motile sperm with good forward motility. The mild oligospermic ejaculate had <30 million sperm per ml. Severe oligospermic ejaculates showed <15 million sperm per ml with less than 40% sperm motility.

Sample Processing:

The samples were processed for sperm concentration analyses using semen diluting fluid. (50gm. of NaHCO3, and 10ml of 35% (v/v) formalin in 1000ml.) The haemocytometer method was used to determine the concentration of spermatozoa 1:20 dilution was made for each sample with diluting fluid 10 (l - 20 (l of diluted samples were used to count the sperms using Neubaur's haemocytometer. The sperms were then counted under phase contrast microscope at a magnification of 200X. Only spermatozoa with morphologically mature germ cells with tails were counted.

Semen homogenisation

The homogenisation of the specimens was done by treatment with tritonX-100. Homogenising butter of 0.1% concentration was prepared using tritonX-100 having pH 7.4.

Homogenising buffer

(11.9 gms of mannitol, 4.8gms of sucrose, 0.09gms of EDTA in 250 ml. of distilled water adjust the pH to 7.4 with tris-base add 250 ml of tritonX-100). The samples were centrifuged at 700 - 80Orpm for 30 minutes and sperm cells separated from plasma 2mi. of the homogenising butter (tritonX-100) was added to the sperm cells and seminal plasma separately and were homogenised. These homogenised samples were used to carry out the tests.

The activity of creatine kinase was measured spectrophotometrically at 340 nm using kits supplied by Boehringer Mannheim Ltd. Also the activity of the isoenzyme CK-MB was measured spectrophoto-metrically using kits supplied by Boehringer Mannheim Ltd[19]. All the chemicals used for the study were of analytical grade. Lipid - peroxidation was studied using method by Ware and Uchiyama[20].

::   Results

[Table:1] depicts the comparison of the water test and lipid peroxidation. Samples which showed > 80 % swelling were considered to be normal and these were compared with others which showed < 40 %, < 20 % and < 60 % swelling respectively. Less the swelling, more was the lipid peroxidation indicating faulty sperm tails. [Table:2] compares the creatine kinase and its isoenzyme creatine kinase MB with the sperm count. Creatine kinase and its isoenzyme showed increased levels in azoospermic and oligospermic patients. This shows a negative correlation with the sperm concentration. [Table:3] depicts the comparison of the creatine kinase and its isoenzymes with the water test. The results show a significant correlation with faulty sperm swelling. [Table:4] compares the lipid peroxidation with the creatine kinase levels. The results indicate that samples from patients with low sperm count and poor swelling of tails show a positive correlation with lipid peroxidation and creatine kinase.

::   Discussion

Water-test and lipid peroxidation were compared in [Table:1] along with the comparison of the cytoplasmic enzyme creatine kinase sperm count [Table:2] and water test [Table:3]. Creatine kinase was also compared with lipid peroxidation [Table:4]. An inverse correlation was observed between sperm count and creatine kinase (p < 0.001). Poorly swollen tails as seen in the water test correlated well with increase in creatine kinase activity (p < 0.001). Lipid peroxidation was also seen to correlate positively with CK activity. Presence of an isoenzyme of creatine kinase CK-MB was also observed in this study.

This isoenzyme was measured spectrophotometrically at 340 nm based on the principle that CK-MB being composed of two moities CK-MM and CK-BB[19].

Creatine kinase molecules are composed of identical subunits (CK-MM or CKBB) or a pair of two different subunits (CK-MB). In mitochondria another CK-dimer (M MI) occurs[21]. In human spermatozoa CK-BB, is localized in the mitochondria of midpiece region by direct immunogold staining[4]. The CK-BB isoenzyme is also found in seminal plasma[9],[10] and this isoenzyme is shown to be several times higher than the levels found in serum, suggesting a local production in male genital system and a multiglandular origin[9].

Claus R et al[22] used a revised extraction method of Huszar et al[21] for separation of seminal CK-BB from spermatozoa to eliminate contamination and increase the yield of creatine kinase extract from spermatozoa. Seminal debris from azoospermic ejaculates showed presence of CK-BB, while the greater portion of isoenzyme separable from CK-MM was CK-Mi from normospermic ejaculates. In the study the presence of isoenzyme CK-MB was observed and was also measured in normal, oligospermia and azoospermia samples and was found to be highest in azoospermic samples.

The CK activity measurements in the direction of ATP synthesis is based on a three step reaction. In the first step, CK catalyses the synthesis of ATP from creatine phosphate and ADP. In the second step the ATP is utilised for glucose-6-phosphate synthesis in the presence of hexokinase. In the third step the glucose-6-phosphate is oxidised to 6- phosphogluconate with reduction of NADP TO NADPH which is measured with an optical density change at 340 nm. Here, the glucose 6-phosphate dehydrogenase (G6PDH) which is an oxido-reductase, calatyses the oxidation of glucose-6 phosphate to 6-phosphogluconate. This step is an important step of the hexosemonophosphate shunt, as it is through this shunt that dihydro nicotinamide adenine dinucleotide phosphate (NADPH) is generated by spermatozoa. This NADPH is the major source of electrons responsible for production of free radicals (O2) by human spermatozoa[23],[24],[25]. it is an important factor for the disruption of spermiogenesis leading to retention of excess of residual cytoplasm by differentiating spermatozoa. Evidence for the hypothesis has come from a number of independent studies indicating that sperm function is frequently associated with elevated activities of certain key enzymes including creatine kinase[2],[12],[13]. These enzymes are not thought to be directly responsible for loss of sperm-function but rather to act as biochemical markers of normality of sperm differentiation. Such errors of spermiogenesis can result in creation of oxidative stress. This could be proved by a positive association between the CK content of human spermatozoa and induction of peroxidative damage[11].

A relationship between incomplete cytoplasmic extrusion and oxidative stress was suggested by Rao et al[26] and others[27] that peroxidative damage to human spermatozoa is associated with morphological abnormalities in the midpiece where excess residual cytoplasm would be localized. The retention of excess of residual cytoplasm may therefore stimulate O2 production through the increasing availability of NADPH as a consequence of enhanced G6PDH activity[28]. A chain of reactions exists involving the retention of excess of residual cytoplasmic enzymes such as CK and G6PDH and enhanced generation of ROS and induction of lipid peroxidation and the appearance of sperm dysfunction. The enzyme creatine kinase was therefore studied in correlation with sperm concentration, which suggested the appearance of impaired sperm function. A remarkable correlation was observed between water test and lipid peroxidation [Table:1] where lipid peroxidation was higher, in less than 60% and 20% swollen samples.

Swelling of sperms is indicative of normal sperm function and transport of molecules across the sperm membrane. Loss of sperm-membrane fluidity due to initiation of lipid peroxidation may cause oxidation induced cellular injury to the spermatozoa[28]. Lipid peroxidation also impairs cell membrane ion-exchange that is essential for maintaining normal sperm-motility[29]. As increase in lipid peroxidation is seen sperm samples with less than 60% and 20% swelling [Table:1]. This is indicative of loss of sperm- membrane fusion events with the oocytes, which is important in the process of fertilization[22]. Semen with low sperm counts showed higher creatine kinase activity [Table:2] indicating that creatine kinase activity may act as a biochemical parameter in detecting sperm quality of the specimen[2]. Higher creatine kinase activity observed in sperm with lesser percentage of swelling [Table:3] indicates defective sperm to be associated with retention of excess of residual cytoplasm. The degree of peroxidative damage sustained by human spermatozoa appears to correlate with defects in the mid-piece of spermatozoa[29],[30]. Higher creatine kinase activity observed with increased lipid peroxidation [Table:4] is indicative of defects in mid-piece with increased lipid peroxidation causing damage to the sperm plasma membrane.

Therefore correlation of water test and lipid peroxidation indicates that water test can be used to screen for sperm membrane integrity. This can also be seen in correlation of water test with creatine kinase activity. A positive correlation between lipid peroxidation and enzyme creatine kinase indicates that tests like lipid peroxidation, and creatine kinase can be used to confirm loss of sperm function. This shows that creatine kinase can serve as a good biochemical marker. Further, water test, which is a very simple test, could be used initially as screening test for sperm membrane integrity and as a potential for lipid peroxidation.

References