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Acetylcholinesterase isoform content and activity in bull sperm during incubation
It is proved, that acetylcholine and acetylcholinesterase (AChE) affect motility and stimulate acrosome reaction in germ cells. In bull semen AСhE activity is provoded by its isoforms, quantity of them can variate from 3–11 of active enzyme proteins. During spermatozoa’s life it metabolizm changes: substrate utilization and ATP resynthesis increases, activates processes of free-radical oxidation.
Purpose of the work is to examine the AChE activity and isoforms during sperm incubation.
Researches were conducted at the Institute of animal biology NAAS and Lviv’s research and production center "Zahidplemresursy". Freshly obtained ejaculates of bull were divided into two parts: one was studied after receiving (fresh sperm), and the rest was incubated for 48 hours in 2–4 ºC. In sperm, plasma and spermatozoa suspension of incubated and freshly obtained semen the total protein content (mg/ml), the AChE activity — during the rate of fast acetylcholine hydrolysis (nmol/min×mg protein). AChE isoforms were detected after electrophoresis in 7,5 % polyacrylamide gel (PAGE) followed by staining with a mixture of alpha-naphthylacetate with blue diazoliyem C. Foregram copies were received by direct PAGE plates scan. Isoforms relative content (%) was calculated using software Soft Spectr 1.3. Isoforms were marked depending on the speed of migration in PAGE — from less to more mobile.
Freshly obtained bull semen contains 51,7±3,52 mg/ml protein and characterizes by AChE activity — 48,3±8,72 nmol/min×mg protein. Also, sperm plasma total protein content and enzyme activity are, respectively, 32,3±0,82 mg/ml protein and 33,7±5,50 nmol/min×mg protein AChE, which is higher than that of sperm 30,6 % (p<0,01) and 44,6 % (p<0,05). In incubated for 48 hours sperm, comparing to freshly obtained, values of total protein are lower on 23,2 % (p<0,05) in semen, on 17,9 % (p<0,01) in plasma and on 41,5 % (p<0,05) in spermatozoa, and the activity of AChE, respectively, on 17,9 %, 13,0 % and 41,2 % (p<0,05).
Thus, bull ejaculates incubation leads to a decrease in total protein content and AChE activity as in sperm and its components — plasma and spermatozoa.
AChE analysis in fresh semen, spermatozoa and plasma indicates unequal number of enzyme isoforms, their different motility in the electric field and shading intensity. In addition, foregrams visual assessment showed that enzyme isoforms main amount and its intensely manifests in semen plasma, lesser — spermatozoa. For freshly obtained bull ejaculates 5-6 major AChE isoforms and 3-4 minor are typical.
Sperm incubation — leads to a decrease in the number of isoforms in 7,5 % PAGE, reducing the intensity of shading and manifestations of active enzyme protein in concentrating gel (3,5 % PAGE ).
By studing relative isoform content in freshly obtained ejaculates it is found that in sperm almost 30,0 % are AChE1, AChE2 and AChE3 content is lower compared to AChE1, respectively, on 12,4 % and 15,0 %, AChE4, AChE6 and AChE7 on 17,9–20,2 % and AChE5 — 23,1 %. The smallest difference between the content of AChE1 and AChE8 — 10,6 %. Unlike, sperm in sperm plasma and spermatozoa AChE4 has the largest number, respectively, 34,1±9,78 and 29,3±1,89 %. The same sperm plasma content (5,0-6,4 %) is of AChE1, AChE2 and AChE7, higher (10,0-11,3 %) of AChE5, AChE6 and AChE8 and even more (17,7 %) — AChE3. In sperm, like plasma, the smallest content of (1,9±0,58 %) AChE1, more (6,6-7,3 %) of AChE6 and AChE7 and highest content of (16,2-18,0 %) AChE3, AChE5 and AChE8.
In the incubated sperm content isoform differs from freshly obtained. Almost 1/3 of the content occupies AChE1 (28,5±7,66 %), 40,9 % — two isoforms AChE4 and AChE5 (18,7±4,95 and 22,2±7,42 %), lesser (4,6-8,0 %) AChE2, AChE3 and AChE6, and the least (3,2±0,36 %) — AChE7. The peculiarity of incubated sperm is the presence of AChE0 (6,9±1,06 %). Unlike sperm, in semen plasma two isoforms AChE4 and AChE5 (respectively, 21,1±4,43 and 25,6±5,16 %) occupy 46,7 % of the total isoform content, AChE1 — 18,8±6,54 %, and AChE2 and AChE6 respectively, 4,8±1,16 and 4,03±0,41 %. In spermatozoa AChE0 and AChE1 occupy 40,9 % (respectively, 16,0±1,26 % and 24,9±2,05 %) of the total isoform content, lesser (16,0-18,6 %) — AChE2, AChE4 and AChE5, the least — AChE6 (6,6±0,72 %). AChE isoform content peculiarity in spermatozoa is AChE3 absence.
Thus, during spermatozoa existence (sperm incubation) reduce not only the activity of AChE, but also changes the number of isoforms of the enzyme.
Key words: acetylcholinesterase, isoforms, sperm, bull.
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