Response of Adnexal Glands to Testosterone Stimulation in the Normal Adult Male

FERTILITY AND STERILITY Copyright c 1976 The American Fertility Society

Vol. 27, No.7, July 1976 Printed in U.SA.

RESPONSE OF ADNEXAL GLANDS TO TESTOSTERONE STIMULATION IN THE NORMAL ADULT MALE FRANCO DONDERO, M.D., DIEGO POZZA, M.D., FERNANDO MAZZILLI, M.D., ALDO ISIDORI, M.D.

AND

Istituto Patologia Medica II, Department of Internal Medicine, University of Rome Medical School, Rome, Italy

Normal male volunteers were studied in order to set up a functional exploration test based on administration of testosterone propionate for use on the adnexal glands of the male genital tract. Seminal acid phosphatase and citric acid levels showed no significant variations compared with spontaneous physiologic oscillations. Concentration and total quantity of fructose were considerably reduced. Alkaline phosphatase showed an intermediate pattern with constant values of concentration and decrease of total quantity. The hypothesis is advanced that adnexal glands possess a secretory metabolism with a maximal basal response to androgen stimulation, but with a different mechanism of action: partly adaptable at the level of the prostate and of the "all or nothing" type at the level of the vesicles.

Seminal plasma is the terminal product of the metabolic processes, partly local synthesis and partly transport, which occur in the adnexal glands of the male genital tract under androgen stimulation. Biochemical analysis of the semen may give a good indication of the endocrine secretion from the gonads and provide useful criteria regarding the functional response of the glands to androgen stimulation. Seminal fluid from normal subjects was investigated in order to observe possible biochemical variations following testosterone stimulation. The aim of the investigation was to establish dynamic criteria of normality as a reference in evaluating the response of the adnexal glands in hypogonadic and/or infertile patients. MATERIALS AND METHODS

The following biochemical evaluations Accepted February 20, 1976.

were made: acid phosphatase, citric acid, fructose, and alkaline phosphatase. Acid phosphatase was evaluated (following dilution of the seminal plasma 1:10,000) with the colorimetric technique using a Boehringer-Mannheim kit; the range of values in normal subjects is 100 to 800 U/ml. 1 Citric acid was evaluated in deproteinized plasma by the pyridine colorimetric method; the normal range is 350 to 670 mg/100 ml.2 Fructose was evaluated with the resorcine colorimetric technique; the normal range 150 to 450 mg/100 ml. 3 Alkaline phosphatase was evaluated with the colorimetric technique using a BoehringerMannheim kit; the normal range is 40 to 190 mU/ml. 4 The study was carried out on 10 volunteers selected on the basis of the following criteria: (1) age between 20 and 35 years and excellent general condition at the time of the study; (2) normal development of primary and secondary sexual charteristics; (3) no disorders in vis and libido; (4) no past history of

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TABLE 1. Concentration, Total Amount, Mean Values, and Percentage Variation of Acid Phosphatase in Basal Conditions and after 15 and 30 Days of Testosterone Administration Acid phosphatase Basal

15th day

30th day

Patient no.

Concentration

Total amount

Concentration

Total amount

Concentration

U/ml

u

U/ml

u

U/ml

1 2 3 4 5 6 7 8 9 10

660 470 210 620 2000 800 930 950 2000 1260

Mean %Variation

990

Total amount

u

2112 1410 588 1736 1800 1680 5580 2755 6400 4410

720 546 210 640 2000 800 1182 840 1920 1515

2808 2129.4 357 1536 2400 1840 6382.8 1596 7296 3939

730 749 180 690 2000 1717 1060 778 1820 2020

2701 2097.2 324 1173 2400 2747.2 2862 1633.8 3640 5252

2847.1

1037.3 +4.77

3028.4 +6.36

1174.4 + 18.62

2483 -12.79

inflammatory or traumatic conditions of the gonads and/or genitourinary tract; and (5) normal pituitary and gonadal function, evaluated by assay of plasma gonadotropins, plasma testosterone, and urinary 17 -ketosteroids, as well as analysis of semen. Semen was collected by masturbation after 3 days of sexual abstinence, stored at 31' C for 30 minutes, and centrifuged at 3000 rpm for 10 minutes. Plasma was then frozen at - 20a C until assayed. Specimens were thawed only at the end of the treatment period, and the individual biochemical substances from each subject, in all samples, were assayed at the same time. Prior to testosterone administration, three specimens of semen were obtained from each subject, once every 3 weeks, in order to establish the spontaneous variations ofthe substances under investigation. In agreement with our previous observations on normal subjects over a period of approximately 100 days, the variations in this group of volunteers were within the following limits: 30% for acid phosphatase, 25% for citric acid, 15% for fructose, and 15% for alkaline phosphatase. Testosterone propionate (25 mg) was ad-

ministered to the 10 subjects on alternate days for a period of 30 days. Semen was obtained under basal conditions (immediately before the first testosterone injection) and on the 15th and 30th days of treatment.

RESULTS

Acid Phosphatase. Acid phosphatase levels during testosterone treatment remained fairly constant. The mean concentration was not significantly modified since the 18% increase on the 30th day was within the range of individual variations. Similarly, the total quantity of acid phosphatase showed no significant variation within the period of time under consideration (Table 1). The 13% decrease on the 30th day, with a simultaneous increase in concentration, was due to the reduction in the seminal volumes: mean basal value, 3.04 ml; 15th day, 2.9 ml (-4.3%); 20th day, 2.22 ml (- 17% ). This decrease after 30 days of testosterone administration, however, was within the range of normal spontaneous oscillations. Citric Acid. The citric acid levels observed during treatment showed

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DONDERO ET AL.

808

TABLE 2. Concentration, Total Amount, Mean Values, and Percentage Variation of Citric Acid in Basal Conditions and after 15 and 30 Days of Testosterone Administration Citric acid Basal

30th day

15th day

Patient no.

1 2 3 4 5 6 7 8 9 10 Mean

Concentration

Total amount

Concentration

Total amount

Concentration

mg/100 ml

mg

mg/100 ml

mg

mg/100 ml

350 230 110 310 1400 470 280 370 610 490

11.20 6.90 3.08 8.68 12.60 9.87 16.80 10.73 19.52 17.15

380 260 110 360 1000 350 320 350 370 490

13.65 10.14 1.87 8.64 12 8.05 17.28 6.65 14.06 12.74

310 410 150 470 1100 600 300 430 410 430

11.47 11.48 2.70 7.99 13.20 9.60 8.10 9.03 8.20 11.18

462

11.65

396 -14.29

10.51 -9.88

461

9.29 -21.26

% Variation

marked oscillations in a few individuals. Mean concentration and total quantity however, were within the range of spontaneous fluctuations (Table 2). Fructose. The decreases in the fructose levels were the most significant finding of the investigation. This reduction was particularly marked in the subjects with highest initial values. The mean concentration showed a reduction on the 15th day (- 17.69%) and remained at these levels on the 30th day (-12.31%) (Table 3).

Total amount mg

Alkaline Phosphatase. The variations in mean concentration of alkaline phosphatase were very slight, whereas the total quantity was considerably reduced on the 30th day (- 22.55%) (Table 4). DISCUSSION

Acid phosphatase, in man, is secreted by the prostate and catalyzes several enzymatic processes, including phosphorylcholine hydrolysis with the formation of free choline and orthophosphate;

TABLE 3. Concentration, Total Amount, Mean Values, and Percentage Variation of Fructose in Basal Conditions and after 15 and 30 Days of Testosterone Adminstration Fructose Basal

30th day

15th day

Patient no.

1 2 3 4 5 6 7 8 9 10 Mean % Variation

Concentration

Total amount

Concentration

Total amount

Concentration

mg/100 ml

mg

mg/100 ml

mg

mg/100 ml

390 230 306 476 14 70 540 236 363 446

12.48 6.90 8.57 13.33 0.13 1.47 32.40 6.84 11.25 15.61

373 170 263 346 52 62 446 270 226 320

307.1

10.89

252.80 -17.69

14.55 6.63 4.47 8.30 0.62 1.43 24.08 5.13 8.59 8.32 8.21 -24.61

363 210 306 423 14 60 418 226 310 363 269.3 -12.31

Total amount mg

13.42 5.88 5.51 7.19 0.17 0.96 11.29 4.75 6.20 9.43 6.48 -40.50

TESTOSTERONE STIMULATION OF ADNEXAL GLANDS

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TABLE 4. Concentration, Total Amount, Mean Values, and Percentage Variation of Alkaline Phosphatase in Basal Conditions and after 15 and 30 Days of Testosterone Administration Alkaline phosphatase

Basal

30th day

15th day

Patient no.

1 2 3 4 5 6 7 8 9 10 Mean % Variation

Concentration

Total amount

Concentration

Total amount

Concentration

mU/ml

mU

mU/ml

mU

mU/ml

98 400 116 123 225 130 45 270 212 100 171.9

316.6 1200 324.8 344.4 202.5 273 270 783 678.4 350 473.97

78 400 88 152 200 130 42 270 150 80

304.2 1560 149.6 364.8 240 299 226.8 513 570 208

159 -7.51

it also takes part in the transfer of phosphate from both phosphoglycerol and phosphocreatine to glucose, with the production of 6-phosphoglucose. 5 In patients with Klinefelter's syndrome, bilateral cryptorchidism, and "Sertoli cells only" syndrome, seminal acid phosphatase levels are statistically significantly lower than those of controls. 6 In normal and hypogonadal males there is a significant correlation between plasma testosterone levels and seminal acid phosphatase concentrations. 1 Androgen administration increases the production of acid phosphatase in hypogonadic patients. 7 Citric acid in man is secreted by the prostate; its presence appears to be correlated to the clotting-lysis mechanism of the ejaculate and to hyaluronidase activity. 8 • 9 Production and secretion of citric acid, as already shown in experimental animals, are androgendependent. 10 This experimental finding forms the basis for the "citric acid test," in which citric acid levels provide an index of testicular secretion of androgens in humans. In normal men and hypogonadic patients, in fact, there appears to be a significant correlation between plasma testosterone levels and seminal citric acid concentrations. 2 Intramuscular

443.54 -6.43

Total amount

mU

94 400 160 120 200 160 52 270 150 80

347.8 1120 288 204 240 256 140.4 567 300 208

168.6 -1.92

367.12 -22.55

administration of 25 mg of testosterone proprionate, once a week for 6 weeks, to patients with oligospermia induced an increase in citric acid levels in those men in whom basal levels were low, and a decrease in those whose basal levels were initially high. 11 Fructose, which in man is secreted by the seminal vesicles via 1-phosphoglucose:6-phosphoglucose:6-phosphofructose, is of prime importance in the metabolism of spermatozoa (prevalently glycolytic, with sugar degradation to pyruvate according to the Embden-Meyeroff scheme) since the spermatic cells which contain very little glycogen depend on seminal fructose. 5 Investigations on experimental animals have demonstrated that fructose secretion is androgen-dependent, and the "fructose test" is also considered a good indication of androgen production in men. 12 - 14 Nevertheless, recent studies appear to exclude any significant relationship between plasma testosterone levels and seminal fructose in normal subjects and hypogonadal males. 3 · 15 • 16 Low seminal fructose levels in the hypogonadal male are seen to increase following the administration of testosterone.17 Oligospermic subjects treated

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DONDERO ET AL.

with testosterone propionate (same mode of administration as for citric acid) showed similar results, i.e., a decrease in high basal fructose values and an increase in low basal values.U An intramuscular injection of 250 mg of testosterone oenanthate once a week for a period of 21 weeks elicits in normal subjects a rapid, significant decrease in the fructose levels, which rise again after suspension of treatment. 18 The response to treatment with 25 mg of mesterolone, three times daily per os for 4 weeks, in subjects with low basal levels of seminal fructose appears to fall into two distinct patterns: type A, in which concentration is increased, is found in Klinefelter's syndrome, male climacteric, and hypogonadotropic hypogonadism; and type B, in which no variation after stimulation and in which aplasia of the seminal vesicles or ejaculate ducts and even prostatovesiculitis may be found, but in which the exact nature of the mechanism failing to trigger a response usually cannot be established.19 Alkaline phosphatase in man is secreted by both the prostate and the seminal vesicles and, like acid phosphatase, plays a role in maintaining a suitable medium for normospermic function. 20 · 21 There does not appear to be a significant correlation between plasma testosterone and seminal alkaline phosphatase levels in normal and hypogonadal males, and low alkaline phosphatase concentrations are hardly changed by the testosterone substitute. 4 The present series of volunteers included only subjects in whom clinical and laboratory tests confirmed a normal andrologic pattern. The aim of the present test with high but not excessive doses of androgens for a reasonably short period of time, therefore, was to establish (1) a simple, functional test to perform in routine clinical practice; and (2) a test which, once standardized in nor-

July 1976

mal subjects and tested in the hypogonadal male, could provide useful criteria concerning the response of adnexal glands during substitution therapy. Testosterone propionate, which we normally use in endocrine substitutional therapy, was employed at maximal maintaining doses. When administered to normal subjects for a period of 30 days, these doses exert a partial and transitory inhibition of pituitary gonadotropin secretion and maintain constant, very high, levels of plasma testosterone throughout the study period. It thus appears possible, without any risk to the patient, to reproduce an efficient and good pharmacologic stimulus that is neither too long nor too intense. In the present studies the modifications in concentration and total quantity of acid phosphatase and citric acid secreted by the prostate are not significant if compared with the spontaneous physiologic fluctuation, whereas the concentration and total quantity of fructose secreted by the seminal vesicles decrease considerably; alkaline phosphatase, secreted at the level of both glands, shows an intermediate behavior, with constant concentration values and reduction of total amount. In the light of data reported in the literature, these results appear to indicate that the adnexal glands show a different response to androgen stimulation. In fact, the prostate, which in hypogonadic males produces low levels of acid phosphatase and citric acid, in normal subjects shows a secretory metabolism with a maximal functional response. Additional administration of testosterone can thus elicit only another partial stimulation. The seminal vesicles, on the other hand, which can produce a normal quantity of fructose even in the presence of androgen deficit-with a similar behavior also as far as ascorbic acid is concerned 22-respond to androgen stimulation with an "all or nothing" response,

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TESTOSTERONE STIMULATION OF ADNEXAL GLANDS

and additional testosterone stimulation in normal subjects results in a kind of functional exhaustion of the gland. Additional factors must also be taken into consideration, i.e., role of gonadotropins and local conversion of blood testosterone into dihydrotestosterone. However, these preliminary findings provide a useful tool in setting up a functional exploration test of the adnexal glands and information on the modality of response of normal subjects to androgen stimulation. REFERENCES 1. Dondero F, Sciarra F, Isidori A: Evaluation of relationship between plasma testosterone and human seminal acid phosphatase. Folia Endocrinol (Roma) 27:185, 1974 2. Dondero F, Sciarra F, Isidori A: Evaluation of relationship between plasma testosterone and human seminal citric acid. Fertil Steril 23:168, 1972 3. Dondero F, Sciarra F, Isidori A: Statistical evaluation of relationship between plasma testosterone and human seminal fructose. Folia Endocrinol (Roma) 23:559, 1970 4. Dondero F, Sciarra F, Isidori A: Relationship between plasma testosterone and human seminal alkaline phosphatase. Folia Endocrinol (Roma) 27:718, 1974 5. Mann T: The biochemistry of semen and of the male reproductive tract. London, Methuen and Co Ltd, 1964, p 182 6. Raboch J, Homolka J: Acid phosphatase in the ejaculate of men with disturbances of somatasexual development. Fertil Steril 12:368, 1961 7. Kent JR, Hill BA, Huix FJ, Segre EJ: Seminal acid phosphatase content in the clinical bioassay of androgens and antiandrogens. Clin Pharmacol Ther 13:205, 1972 8. Huggins C, Neal W: Coagulation and liquefaction of semen. Proteolytic enzymes and citrate in prostatic fluid. J Exp Med 76:527, 1942

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9. Baumberger JP, Fried N: Magnesium as an activator of antinvasin. J Biol Chern 172:347, 1948 10. Humphrey GF, Mann T: Studies on the metabolism of semen. 5. Citric acid in semen. Biochem J 44:97, 1949 11. Lavieri JC, Calamera JC: Effects of gonadotropins and androgens on fructose and citric acid of seminal fluid. In The Human Testis, Edited byE Rosemberg, CA Paulsen. New York, Plenum Press, 1970, p 515 12. Mann T, Parsons U: Effect of testicular hormone on the formation of seminal fructose. Nature 160:294, 1947 13. Landau RL, Loughead R: Seminal fructose concentration as an index of androgenic activity in man. J Clin Endocrinol Metab 11:1411, 1951 14. Kimmig J, Steeno 0, Schirren C: Ergebnisse der modernen Biochemischen forschungen auf dem Gebiete der Andrologie. Internist (Berlin) 8:25, 1967 15. Moon KH, Osborn RH, Yannone ME, Bunge RG: Relationship of testosterone to human seminal fructose. Invest Urol 7:478, 1970 16. Moon KH, Bunge RG: Seminal fructose as an indicator of androgenic activity: critical analysis. Invest Urol 8:373, 1971 17. Cassano C, Conti C, Andreani D: La Sterilita Endocrina Maschile. Livorno, Italy, Stabilimento Poligrafico Belforte, 1953, p 183 18. Borsch G, Mauss J, Leyendecker G, Nocke W, Richter E: Depression of human seminal vesicle function during log-term administration of testosterone. J Reprod Fertil 41:219, 1974 19. Mauss J, Borsch G, Torok L: Differential diagnosis of low or absent seminal fructose in man. Fertil Steril 25:411, 1974 20. Bern HA: The distribution of alkaline phosphatase in the genital tract of the male mammals. Anat Rec 104:361, 1949 21. Moon KH, Bunge RG: Observations on the biochemistry of human semen. II. Alkaline phosphatase. Fertil Steril 19:766, 1968 22. Dondero F, Vasselli C, Sciarra F, Isidori A: Ascorbic acid in human seminal plasma in normal and pathological conditions. Folia Endocrinol (Roma) 27:724, 1974