Cytochemical study of ejaculate

Cytochemical reaction, by èâkulâtom, allows to judge about the chemical composition and its functional activity of cells.

Determination of the acid phosphatase activity of ejaculate

The activity of acid phosphatase in the ejaculate is many times greater than its activity in the blood. Acid phosphatase is a part of the prostate gland, products it is stimulated by male hormones gonadotropnymi. Using cytochemical reactions she detected in sperm and other cells in ejaculate. Its activity is defined a method Goldberg-Barca.

Reagents.

1. Formalin alcohol (one part of the finished 40 % formalin dissolved in nine parts 96% ethyl alcohol).

2. Pararozanilin: 1 g basic fuchsine for Fuk- sinosernistoj acid is dissolved when the warming in the 25 ml 2 n hydrochloric acid; cooled, filtered and stored in the refrigerator. Reagent resistant.

3. An aqueous solution of sodium nitrite 4 %. 4. Solution of methyl green to acetatnom buffer 2 % (pH — 5). The resulting solution washed chloroform, Why mix approximately equal amounts of chloroform and 2 % solution of methyl Green. Shake up many times during the day, and then okrasivshijsja in purple chloroform Removed by using separating funnel.

5. Incubation Wednesday, consisting of two components.

• And component: 20 mg-A5-naphthol phosphate dissolved in 0,5 (0,3) ml of dimethylformamide, add 40 ml 0,1 n solution of sodium acetate and filtered through filter paper; prepare daily studies.
• B-component: 8 drops 4 % sodium nitrate solution was mixed with 8 pararozanilina drops; cooked ex tempore. To get both components Wednesday incubation connect (pH 5.2-5.4). If necessary pH correct either 50 % acetic acid, either a solution of caustic alkali.

Method. Fresh ejaculate centrifuge, When a large number of sperm without centrifugation. Of the sludge cooked cut glass thin smears, that, like blood spots, dried in air. Further dried smears record by dipping them in a cup with formalinovym alcohol on 30 from (no more) and washed with distilled water. Then swabs dipped in a cup with incubation Wednesday and put in a thermostat at a temperature 37 ° C 2 no. Then rinse them with distilled water, immerse the 10 mines in the Cup with a solution of methyl Green (for dokrashivanija cores), quickly wash water, dried filter paper and explore using immersion microscope system.

Acid phosphatase activity can be expressed using the average citohimicheskogo ratio (SCK) by polukolichestvennomu method Astal'di-Verga. According to the formula

SCK =(3*and +2 * b +1 *)/100

where numbers in the numerator indicate intensity of coloring (maximum - 3, moderate — 2, traces- 1), and letters — the percentage of cells counted a certain intensity of colouring; figure 100 the denominator is the total number of cells counted.

For Example, regarded 100 cell, of them with a maximum intensity of coloring 20 cell, with moderate- 20 and with traces of — 60 cell. From here:

SCK =(3*20+2*20+1*60)/100= 1.6

Spermatozoid in acid phosphatase is located in the form of a single large granules of red-orange in the top department heads.

Taken by chromatin of the head is green and contains no acid phosphatase.

More pale diffuse coloring on acid phosphatase is detected around the bottom of the head, and sometimes extends to the neck of a spermatozoon. Average coefficient of cytochemical acid phosphatase in normal sperm ejaculate is 2.0-2.4.

In the cytoplasm of immature germ cells are detected by the accumulation of granules, bright okrashivajushhihsja in specific (red-orange) color. In the supporting cells of convoluted seminiferous tubules, often dramatically stained positively for acid phosphatase, prosvechivajutsja unpainted heads (xromatin) sperm. Histiocytes and macrophages contain few granules, positively stained by acid phosphatase. In isolated lymphocytes, and sometimes the neutrophil granoulozitah meet individual granules, the nuclear activity of acid phosphatase.

Determination of activity of succinate dehydrogenase (SDG) in the ejaculate

Sukcinatdegidrogenaza - Enzyme, involved in redox reactions, contained in the cells of the ejaculate in a significant number of. However, the nature of its activity changes in various pathological processes is not yet well understood. The activity of succinate dehydrogenase in cells is determined by the modified Narcissova method.

Reagents.

1. Acetone-trilon (acetone is 60 ml, distilled water is 40 ml, Trilon b 250 mg).

2. Solution of methyl Green 2 % (the same dye, which is used for acid phosphatase).

3. Incubation Wednesday, consisting of the following components::

and) phosphate buffer (pH 7.2-7.4) - 10 ml;

to) 5,4 % solution of sodium succinate- 10 ml;

in) trilona b (13 mg diluted in 5 ml distilled water);

g) distilled water is 5 ml;

d) nitrotetrazolija purple (10 mg diluted in 10 ml of water).

All the components are mixed in one bottle and store in the fridge. Finished Wednesday may be used multiple times within 10 days or more.

Method. The strokes, made from ejaculate, fixed in acetone-trilone during 30 from, washed with distilled water and dried in the open air. Then swabs dipped in incubation Wednesday and put in a thermostat at a temperature 37 ° C 1 no, then washed with distilled water and dokrashivajut methyl green during 10 m. Next again quickly washed with water, dried filter paper and explore using immersion microscope system.

Normal sperm have a high activity of succinate dehydrogenase, submitted by large blue-purple granules, that, merging between a, form a dense mass, fill neck sperm, some sperm individual granules are found around the head.

Calculate the fusion with each other pellets is not possible, Therefore, to determine the activity of succinate dehydrogenase painted part of sperm measured in micrometers. On length specific painted on the sukcinatdegidrogenazu part of the sperm head is judged on the degree of activity of the enzyme in this spermatozoide. Using an eyepiece micrometer the micrometer and measured length specifically painted parts in 100 sperm. Folded length stained plots counted spermatozoid and dividing the resulting figure by 100, get the average length, expressing sukcinatdegidrogenaznuju activity for one sperm. OK it is 4.3 ± 0.7 μm. This counting activity SDG most informative and is therefore recommended for use for scientific purposes.

For wide application in practice this method is somewhat complicated, It is preferable to express the number of succinate dehydrogenase in Astal'di method-Verga. Average coefficient of cytochemical SDG sperms in a normal ejaculate is 2,7 ± 0.3.

In cells of spermatogenesis SUCCINATE DEHYDROGENASE activity is detected in the form of clusters of large blue-purple granules, often merging into larger lumps, located in different parts of the cytoplasm. Average coefficient of cytochemical in cells of spermatogenesis does not reach 2,0. As the maturation of cells activity increases and SDG ejaculate in mature sperm exceeds 2,0. In the cytoplasm of certain lymphocytes discovered large granules SDG.

Determination of peroxidase activity in the ejaculate

In sperm, cells of spermatogenesis and the supporting cells of convoluted seminiferous tubules peroxidase is missing.

In the neutrophil granoulozitah ejaculate peroxidase activity expressed in heavily and may vary depending on the nature of the pathological process, as well as in the peripheral blood. The level of activity of peroxidase in the neutrophil granoulozitah is important for assessing the functional status and inflammatory activity in the male genital organs. Coloring the stroke by Graham Knollju-ejaculate and accounting activity of an enzyme by Astal'di is similar to blood strokes held Vergu.

Peroxidase activity in the cells of the ejaculate is determined by Graham's method — Knollja.

Reagents.

1. Formalin alcohol (same, as for acid phosphatase).

2. Peroksidaznyj reagent: ʙenzidin (at the tip of the scalpel) dissolved in 6 ml 96 % ethyl alcohol, add 4 ml of distilled water 0,02 ml 3 % hydrogen peroxide. Reagent is fit to eat within 5-6 days.

3. Dye Romanowski in breeding 1-2 drops on 1 ml distilled water.

Method. Fresh ejaculate strokes record during 30 with formalinovym alcohol, washed with running water and dry up. Then pour the reagent peroksidaznym strokes 5-7 min, washed thoroughly in running water and dry up. After their 15-dokrashivajut 20 min dye Romanowski, washed with running water, dried and mikroskopirujut using immersion microscope system.

With the positive reaction to the peroxidase in neutrophil granoulozitah detected large golden-brown granules in a significant number of. When a large number of cells you want to count average coefficient of cytochemical (SCK). In the cytoplasm of histiocytes contain peroxidase isolated in small clusters of the same pellets.

Determination of glycogen in ejaculate

Content changes of glycogen in different cells affect their function or attest to the development of pathological process. Determination of glycogen in cages shall be carried out Mack Manus method.

Reagents.

1. An aqueous solution of iodine acids 1 % (preparing to use).

2. Sulphurous water: 5 ml 10 % as metabisulfita potassium or sodium, 5 ml 1 n hydrochloric acid, water up to 100 ml (preparing to use).

3. Schiff Reagent: 1 g basic fuchsine for fuksinosernistoj acid, pre pounded in a porcelain mortar, dissolve with constant shaking during 5 min in 200 ml of boiling distilled water, pass through a paper filter and add cooling to 50 ° C 20 ml 1 n hydrochloric acid, and when cooling down to 25 ° C is 1 g as metabisulfita potassium or sodium. Prepared reagent stand in the refrigerator for 24 h after which he clarifies and Stano families fit for human consumption

4.Aqueous solution of methyl Green 2 %, washed chloroform

5.Chemically pure methyl alcohol.

Method.

Thin dried smears ejaculate fix chemically pure metilovam alcohol during 3 m, rinse with distilled water and dipped in a cup with 1 % an aqueous solution of iodine acids on 5 m. Then smears are washed with distilled water, dried, rinse in sulphurous water and dried again.

Further down strokes in glass with Schiff reagent on 15 m, Rinse them in three portions of the sulphurous water (by 3 min each), wash out 10 min in water and dokrashivajut nuclei 3 m methyl Green. Then opolascerve water strokes, dried filter paper and mikroskopirujut using immersion microscope system.

In mature sperm glycogen is missing. In the cytoplasm of young sperm, especially with the "collar", detected traces of glycogen (±), sometimes in the form of individual granules at the top of the head or in the collar ".

In cells of spermatogenesis glycogen is found in large quantities in the form of cherry-red granules, populate all cytoplasm. As the maturation of cells lose their glycogen until his disappearance in mature sperm. Spermatotsity contain glycogen not only in the cytoplasm, but in the kernel. In the supporting cells of convoluted seminiferous tubules glycogen is positioned as a cherry-red granules and diffusive. Mature neutrophil granulocyte in ejaculate, as in peripheral blood smears, contain significant amounts of glycogen. If Chronic prostatitis in neutrophil granoulozitah ejaculate is greatly increased glycogen and peroxidase activity. In isolated lymphocytes normally sometimes glycogen granules.

Macrophages contains traces of glycogen Von smears ejaculate turns cherry red, that suggests a large amount of ejaculate plasma glycosaminoglycans (mukopolisaxaridov).

Determination of ribonucleic acid (RNA) in the ejaculate

Elevated levels of RNA in cells evidence of high activity on growth, secretory, synthetic and other activities. Reduction of RNA in young cells often associated with decreased the ability of these cells to regenerate. Content of RNA in cells is determined by the Kurnika method.

Reagents.

1. Kurnika Reagent: prepare separately 2 % solutions of methyl green and pironina, solution of methyl green thoroughly washed with chloroform; work the dye solution prepared by mixing 2 % solution pironina (12,5 ml) and 2 % solution of methyl Green (7,5 ml) with the addition of 30 ml distilled water. Stain resistant, stored in the refrigerator.

2. The retainer is chemically pure methyl alcohol.

Method. Fix strokes metilovam alcohol during 3 m, paint the working solution of pironin-methyl Green 15 m, fast opolascerve water and dried filter paper. Mikroskopirujut using immersion microscope system. RNA turns pyronin in bright red color, the nuclei — methyl green to green.

Mature sperm cells do not contain RNA. Cells of spermatogenesis are rich in RNA, amount of which decreases in cells as their maturing. From spermatogony cytoplasm rich pink color, from spermatocitov It is paler, and do spermatids pale-pink. The young have sperm "collar" Pink painted predominantly "collar".

Sperm, are in the final stages of its development, in support of convoluted seminiferous tubules cells lose the pink residue substance (RNA) and grow into mature sperm.

Immature germ cells contain large amounts of glycogen, RNA and much activity expressed and KF SDG. But, usually, These single cells in the ejaculate, so counting SCK design. In such cases shall be limited to the narrative form of conclusion.

Cytochemical characteristics of leukocytes an important indicator of their functional activity. The number of substances in leukocytes ejaculate just as, as in other cells and peripheral blood in ejaculate.

In conventional smears stained ejaculate (azurozinovymi mixtures, haematoxylin-eosin, etc.), as well as in native medicines are not always easy to distinguish between leukocytes, cells of spermatogenesis, the epithelium of the prostate, histiocytes and other elements.

Cytochemical methods in conjunction with morphological often help set the kind of cells. So, expressed as peroxidase activity, provided by the large golden-brown granules, filling all the cytoplasm of the cell, characteristic of mature Neutrophilic granulocytes, other cells this response does not occur. Expressed reaction on glycogen is typical of Neutrophilic granulocytes and cells of spermatogenesis, but the latter do not have peroxidase activity. RNA is contained in cells of spermatogenesis and absent in mature granoulozitah. Cells of spermatogenesis have pronounced reaction to the acid phosphatase, and in neutrophil granoulozitah it expressed very poorly, and so. d.