1. Methods characterizing the stimulating effect on spermatogenesis

Spermatogenesis is a complicated process, covering proliferation of the spermatogonia, long-lasting process of the tissue meiosis and numerous changes in the spermatids during their preformation. The effect on the sexual cells can occur during the reproductive period - mitotic division of the spermatogonia or during the maturation of the spermatozoa. The effect on Tribestanol on mitosis and maturation of the gonocytes has been studied using quantitative cytological methods. After oral administration of Tribestanol in a single daily dose of 70 mg/kg body mass for 20 days, the testes of 8 rats were fixed in neutral formol-calcium and in Serra's solution, and later embedded in paraffin. The testes of 8 untreated animals were used as control. The histological preparations from the testes were stained with hematoxylin (after Mayer) and fast-green (after Yordanov, 1976). Spermatogonia, spermatocytes and spermatids of 40 cross-sections through the seminiferous tubules were counted for each animal from both experimental and control groups (a total of 640), with identical diameter of the tubules (determined by eyepiece micrometer) in phase VII, according to the classification of Leblond and Clermon (1952).
Using light microscopy, the thickening of the layer of the spermatogenesis cells was observed in the cross-sections of the seminiferous tubules and a narrowing of their lumen in the treated animals. That resulted from the increased number of rows of sexual cells (Fig. 1). The number of spermatogonia in the 8 experimental animals (i.e. in 320 sections of the seminiferous tubules) was 58 spermatogonia on the average per seminiferous tubule (between 48 and 63). The number of spermatogonia in one seminiferous tubule in the control animals was 36 (between 36 and 40 spermatogonia per tubule). The mean number of spermatocytes in a seminiferous tubules was identical to that of the spermatogonia. The number of spermatids in phase VII varied from 148 to 180 per seminiferous tubule in the treated animals (mean value 176). Their number in the control animals was between 112 and 125 (mean 119). The preparation significantly increased the number of spermatogonia, spermatocytes and spermatids in the testes of rats, with no other effect on the diameter of the seminiferous tubules.
Figure 1. Stimulating effect of Tribestanol on spermatogenesis

2. Effect on DNA synthesis in gonocytes

The preparation's effect on DNA synthesis in the sexual cells has been studied by cytohistoradiography. The testes of rats treated with Tribestanol (for 7 days) and with 3H-thymidine (every second day), and later with colchicine (3 hours prior to decapitation), were fixed in Serra's solution and embedded in paraffin. The sections were covered with Ilford liquid emulsion and left to stay for 25 days. A higher number of 3H-thymidine-labelled spermatogonia type "A" and "B" was found in the treated rats compared to the control animals.
The mean number of spermatogonia per section from the seminiferous tubules was 56 in the treated animals, 41 of them labeled with radioisotopes. These numbers were 50 and 18 respectively, in the control animals. The increased number of spermatogonia, with 3H-thymidine included for the treated animals, suggested an intensified DNA synthesis under the effect of Tribestanol, as well as an increased number of spermatogonia during the phase V of the cell cycle.

3. Effect on Leydig and Sertoli cells in the testes

It is well known that Leydig and Sertoli cells participate in the process of spermatogenesis. Quantitative cytological methods were used for the evaluation of the effect of the Tribestanol on these cells. The results show that the number of Sertoli cells was increased in the seminiferous tubules of Tribestanol-treated animals, compared to the controls.
The mean number of Sertoli cells in a section of the seminiferous tubule in the treated animals was 29 versus 19.50 in the controls (increase by 40%). The cytological studies of the testes showed no differences in the number of Leydig cells between the experimental and control animals.



4. Effect on concentration, motility and survival of spermatozoa

The concentration, motility and viability of spermatozoa in the epididymis of rats treated for 30 days with Tribestanol were studied immediately after decapitation. Sodium citrate was used as diluent. The mean spermatozoa number per ml was higher by two million in the treated animals, compared to the controls.
The number of motile spermatozoa under the microscope was 8% higher in the treated animals. Furthermore, their spermatozoa were more viable. The loss of their advancing movements could be observed on the 75th minute, on the average, and in the control animal group - by the 45th minute.


5. Effect on the sexual libido

The effect of Tribestanol on the sexual behavior was studied on male pigs with confirmed lasting impotence. The preparation was administered orally and its effect on the sexual behavior and sexual reflexes was followed up daily. Individual animal reaction to the preparation was observed. The libido and sexual reflexes were restored in 71% of the animals with complete absence of libido, treated with a daily dose of 70 mg/kg for 10 days. In the animals with poor libido and long reflex period of sexual reflexes, recovery was recorded in100% of the cases.

Studies on serum concentration of the hormones from the hypophyseal-gonadal axis

The experiments were carried out on healthy subjects (8 male and 8 female), aged between 28 and 45 years (Milanov et al., 1981). The preparation was administered orally in a dose of one capsule, three times daily at 8-hour intervals for 5 days. The basal hormonal levels were determined before and after the intake of the Tribestanol (at 8:00 am and at noon). The concentrations of the luteinizing (LH) and follicle-stimulating (FSH) hormones were determined by kits provided by Biodata (Italy). Serum testosterone was determined by the method of R.H.Williams (1967), serum estradiol - by the method of C.P.Orezyk (1974), using kits provided by the Sorin (Belgium). The results reveal that the drug elevated the level of the luteinizing hormone and testosterone in the orally treated healthy males, not affecting FSH.
In the females, the concentration of FSH and estradiol were increased under the effect of Tribestanol, whereas the testosterone concentration was not significantly changed. The results show that the preparation has an effect on the hormones from the hypophyseal-gonadal axis, while at the same time not disturbing the hormonal balance in the body, thus enabling its administration as an agent stimulating the reproductive function.


7. Effect on the central nervous system

The screening system for neuro-pharmacological tests (R.Nikolov, 1980) was used in the studies. The following parameters of the treated animals were observed during the first stage of the screening: awareness, mood, motor activity, muscle tone and somatic reflexes.
The second stage of the screening covered the administration of many substances with an effect on the central nervous system, e.g. corazol, strychnine, nicotine, arecoline, phenamine, sodium hexobarbital, reserpine. The drug was applied itraperitoneally to albino mice, H line, with a body mas of 18 - 22 g.
With a dose of 100 mg/kg body mass (1/4 of LD50), the drug had no effect on the behavior of the contact animals in the cage. During observations out of th cage, the animals became more excited, with enhanced reactivity. Their muscle tome was simultaneously reduced. In that dose, the drug inhibited moderately the corazol-induced convulsions, but the other reflexes were suppressed. The maximum tolerance dose - 300 mg/kg body mass - led to reduction of the motor activity, slight disturbance of gait and lower muscle tome of the limbs and stomach.

8. Effect on the cardiovascular system

The effect of the drug on the blood pressure values of cats under urethan narcosis was studied by the method of Ludwig Zyon (S.Vankov, 1981). The drug was injected intramuscularly and itraperitoneally as 10% aqueous solution. The intramuscular application of the drug in doses of 50, 100 and 150 mg/kg body mass had no significant effect on the blood pressure of the urethanized cats. A significant hypotensive effect was observed with the intraperitoneal application of the drug in a dose of 150 mg/kg body weight, advancing from the 5th to the 10th minute after application. The values of the arterial pressure decreased by 20% compared to the initial ones. The oral administration of Tribestanol in a dose of 150 mg/kg on awake dogs had no effect on the blood pressure. The oral administration in doses of 50, 100 and 150 mg/kg body mass had no effect on the autonomic nervous system of the urethanized cats.

9. Pharmacokinetic studies

The experiments were carried out on albino, Wistar rats (180 - 200 g body mass) in 1981 by N.Dikova and V.Ognianova. the unchanged protodioscine in plasma, bile and urine was measured by thin-layer chromatography. Semi-quantitative measures were recorded, standardized by the precisely determined protodioscine concentrations. To determine the concentration of plasma protodioscine, the animals were intravenously injected single doses of 50 and 200 mg/kg body mass. Citrate blood was withdrawn 2, 4, 10, 20, 30, 45, 60, 90, 120 and 180 min after injection. To determine protodioscine excretion in the bile the animals were treated intravenously and orally with single doses of 50 and 200 mg/kg.
The bile was dynamically collected: up to the 6th hour, from the 6th to the 9th hour, from the 9th to the 24th hour after each application. Twenty-four-hour urine was collected. The results show that protodioscine was rapidly eliminated from the plasma and its concentrations were insignificant after the 180th minute. About 12 to 14% protodioscine were excreted in the bile and about 6 - 7 % in the urine within 24 hours after the intravenous administration of the doses of 50 and 200 mg/kg. Protodioscine from 2 to 4% were excreted with the bile after oral administration. No measurable concentration of unchanged protodioscine was found in 24-hour urine after oral administration.

10. Toxicological studies (G.Tanev, S.Zarkova, 1980)

10.1. Acute toxicity
The acute toxicity of Tribestanol was studied after intraperitoneal and oral application to albino mice, H line (18 - 20 mg body mass) and albino rats (160 - 180 g body mass). LD50 was also studied. It was concluded that the product can be included in the group of practically non-toxic substances. LD50 was 1942 mg/kg body mass with intraperitoneal application to mice and over 10,000 mg/kg body mass - with oral administration. The mean lethal dose of Tribestanol with intraperitoneal application to rats was 750 (375 +/- 1,500 ) mg body mass, and after oral administration - over 10,000 mg/kg.
10.2. Subacute toxicity
The Tribestanol was administered orally to albino Wistar rats for 30 and 90 days in the following doses: 75 mg/kg, 150 mg/kg, 225 mg/kg and 300 mg/kg body mass. No increased lethality was observed, nor a change in the behavior of the animals. No significant changes were observed in the routine clinical-laboratory and biochemical indices, nor morphological changes in the internal organs.
10.3. Chronic toxicity
Tribestanol was administered orally to albino rats for 6 months in doses of 75 mg/kg and 150 mg/kg body mass, as well as in 75 mg/kg body mass for 180 days to Beagle dogs. The following toxic symptoms were looked for: changes in behavior, changes in the hematological, biochemical, functional and morphological parameters. No significant changes were found both in the behavior and in the reflexes of the animals. No increased lethality was observed. No pathological deviations from the physiological values were found in all hematological and clinical-chemical indices studied. No pathological changes in the structure of the internal organs, related to the toxic effect of the preparation, were detected.
Teratological and embryotoxic studies were simultaneously performed, as well as some experiments to follow the pre- and postnatal development (Z.Ilieva, 1980).
No teratogenic and embryotoxic action, nor deleterious effect on the development of the first generation after its littering, were found after the oral administration of the product in a dose of 750 mg/kg body mass to pregnant Wistar rats.
Studies were carried out to exclude the possible carcinogenic potential of Tribestanol during a long-term treatment of rats (Gendzhev, 1981).
Increased incidence of neoplasms compared to the control animals was not observed with daily doses of 50 and 150 mg/kg body weight, administered orally for 23 months. No toxic damage was found morphologically in the rat organs.

11. Discussion of the results

The experimental data on the biological activity of Tribestanol show that its oral administration to rats significantly increased the number of spermatogonia, spermatocytes and spermatids, without any changes in the diameter of the seminiferous tubules. This fact is associated with the confirmed stimulating effect on spermatogenesis as a whole. It is well known that DNA synthesis occurs in the s-phase of the mitotic cycle. A fact of certain interest is that a significant increase of type A and B spermatogonia was found in the rats simultaneously treated with Tribestanol and 3H-thymidine during the s-phase.
Hence, it can be concluded that the product intensifies the mitotic activity of spermatogonia. The cytologically detected increased incidence of Sertoli cells, caused by the product, presupposes that the mitosis of these cells has also been stimulated. The important role of Sertoli cells in the regulation of spermatogenesis is well known (Lacy, 1967; Kerr and Klester, 1974, Steinberger, 1971), hence the increased number of Sertoli cells during Tribestanol treatment should be associated with the intensification of spermatogenesis. No changes were identified in the Leydig cells of the experimental animals, which suggests that the effect of the product on the spermatogenesis probably does not include these cells. The literature data show that the proliferation of spermatogonia in mammals and birs is FSH-stimulated (Stoinberger et al., 1964; Mancini et al., 1966; Ishiis and Furua, 1975; Krueger et al., 1974). The authors presume that the effect of FSH on spermatogenesis is due to Sertoli cells. The radioimmunological studies on healthy males showed no changes in the FSH-level under Tribestanol effect, which suggests presence of a selective effect of the product on gonocytes. On the other hand, elevated LH-levels were found in Tribestanol treated healthy males, which suggests the existence of central action.
The pharmacokinetic studies reveal no measurable concentrations of the product in the plasma after oral administration to rats, but spots unidentified so far were detected by the chromatographic methods.
The authors (Dikova and Ognyanova) presume a biotransformation of the product in the body. In such cases, some of the metabolites formed during the biotransformation can be expected to possess a stimulating effect at hypothalamic level.
The effect on the libido of the male pigs is clearly manifested. Tribestanol not only stimulates the libido, but also possesses a therapeutic effect as well in the cases of impotence, manifested in complete absence of libido. The effect of the product on the quality of the spermatozoa clearly shows that the spermatozoa of the treated animals are more viable and more resistant, suggesting a better fertility. Many researchers believe that the sexual behavior of the animals and the motility of the spermatozoa depend on testosterone levels. Other authors think that the sexual behavior is modulated by dehydrotestosterone. The problem of the mode of modulation of the sexual behavior remains debatable. If we assume that androgen-like factors are formed through biotransformation in the body, they would not induce changes in the interstitial cells.
Special attention should be paid to the harmlessness of the product. No evidence of acute, subacute and chronic toxicity has been found during the experimental behavioral, hematological, functional, biochemical and morphological studies. No data on carcinogenic and teratogenic effect are available.
The fact that the product has an effect on the hormonal balance in the body, without disordering its regulatory mechanisms, is of equal importance. The combined action of the drug (stimulation of sexual libido and spermatogenesis) and the absence of adverse effects, characterize it as an original agent for the treatment of males with disordered sexual function.