Acrosin is a trypsin – like serine proteinase found within the acrosome of human spermatozoa has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality.
Spermatozoa are particularly susceptible to OS – induced damage because their plasma membrane scontain large quantities of polyunsaturated fatty acids that are required to maintain the membrane fluidity. This fluidity regulates specific functions such as acrosome reaction and fusion with oocyte membrane. The final endproduct of lipid peroxidation, malondialdehyde, estimated by thiobarbituric acid (TBA) assay which can be used to gauge the level of OS in semen. Coenzyme Q10, a component of the mitochondrial respiratory chain, also known as ubiquinone for its wide diffusion throughout mammalian tissues, plays a key role in energy metabolism and has potent antioxidant properties for cellular membrane integrity.
Ubiquinone could cover important functions in seminal fluid, due to its metabolic and antioxidant properties. In fact, it is known that a large amount of mitochondria is present in spermatozoa, in which the motile activity requires a high energy expenditure. Objectives: To study the association between semen oxidative stress, CoQ10 concentration (by improved HPLC method) acrosin level and enzymatic profile with semen parameters and sperm acrosomal status (by new visualization method) in different male infertility types and healthy controls. Subjects and methods: Sixty male patients with infertility were recruited Al – Kadhimia teaching hospital – Baghdad and included in this study from November 2004 to July 2006. The patients were categorized according to their seminal fluid parameters to oligozoospermia (n=32), azoospermia (n=22) and asthenozoospermia (n=6). All results obtained were compared with age – matched healthy controls group consisting of 39 subjects. The experiments were carried out in the laboratories of the physiological chemistry department at the college of Medicine / Al – Nahrain University. Coenzyme Q10 levels were measured by improved isocratic reversed phase HPLC technique with UV detection at 275 nm.
The seminal fluid acrosin levels were estimated by an improved spectrophotometric method whereas density gradient centrifugation technique was used in the assay to separate sperms from the seminal plasma with a good quality.MDA levels were quantified by a spectrophotometric method depends on the reaction between malondialdehyde elaborated as a result of lipid peroxidation with thiobarbituric acid forming a pink color which measured spectrophotometrically at 534 nm. total creatine kinase activities were measured kinetically by monitoring the NADPH production at 340 nm whereas MB isoenzyme activities were measured using monoclonal antibodies directed against the CK-M subunit provoking complete inhibition of CK-MM and partial inhibition of CK-MB. Determination of the residual activity (CK-B subunit) enables calculation of CK-MB activity in the sample. Results: The mean seminal plasma CoQ10 was 1.10±0.169 mg/L in oligozoospermia, 0.567±0.098 mg/L in azoospermia, 0.740±0.06 mg/L in patients with asthenozoospermia and the control level was 1.652±0.139mg/L. The seminal plasma CoQ10 levels in all infertility groups showed a significantly difference from the control group (P≤0.0001). High significant decrease (P≤0.0001) of the acrosin activity was found in the seminal fluid of oligospermic men (n=32) 22.542±2.534 μIU/106 sperm and asthenospermic men (n=6) 19.315±2.419 μIU/106 sperm from the mean control group (n=39) level 41.69±3.122 μIU/106 sperm. High significant increase (P≤0.001) in the MDA levels was noted in the seminal plasma of oligozoospermia 11.37±1.64 µmole/L, azoospermia 13.87±1.62 µmole/L and asthenozoospermia group 9.508±0.533 µmole/L whereas the level in the control group was 8.517±0.622 µmole/L. Acrosomal integrities of spermatozoa were evaluated by an improved staining technique. The (mean ± SD) of the acrosomal integrity percents of oligospermic patients was 60.65±4.53% whereas in asthenospermic patients the mean value was 61.00±1.67%. On the other hand, the acrosomal integrity percent in the fertile control subjects was 65.41±5.62%. Total creatine kinase activities showed very high significant elevations (P≤ 0.001) in all infertility groups. Seminal plasma total creatine kinase activities were 313.485±19.12 U/L in oligozoospermia group, 451.67±41.30 U/L in the asthenozoospermia group and 585.72±21.87 U/L in azoospermia group. Whereas the seminal plasma total CK level in control group was 274.808±19.64 U/L. On the other hand, seminal plasma CK-MB isoenzyme levels were 65.63±4.92 U/L in oligozoospermia group, 89.123±12.38 U/L in the asthenozoospermia group, 123.192±2.63 U/L in azoospermia group and 50.457±3.53 U/L in the control group. Arylsulfatase A and B were significantly decreased in all infertility groups. Seminal plasma arylsulfatase A decreased 73.55% in oligozoospermia group, 75.14% in azoospermia group and 73.87% in asthenozoospermia group in relation to the normal fertile group. Seminal plasma arylsulfatase B decreased 72.78% in oligospermic patients, 22.34% in azoospermic patients and 44.82% in asthenospermic patients compared with the normal fertile group.
Seminal plasma NAGase level was markedly low (74.44%) in azoospermia group while it was nearly unaffected in oligozoospermia and asthenozoospermia groups (6.76%, 2.04%) respectively. On the contrary, seminal plasma alkaline proteinase increased 1.55, 1.86 and 2.11 fold in oligospermic, azoospermic and asthenospermic groups respectively compared with normal group. Seminal plasma CoQ10 was inversely and significantly correlated with MDA(r=- 0.760; P= 0.000). In addition, seminal plasma MDA correlated significantly and positively with CK-MB (r= 0.744; P= 0.000). Conclusions: A new improved HPLC method for the quantitation of CoQ10 is suitable for analysis of blood plasma, seminal plasma and sperm extract samples. It is possible to use seminal fluid acrosin level and seminal plasma CK-MB as fertility markers with different interpretations.