- Email: [email protected]
A Direct Effect of Antiovulatory Compounds on Human Testicular Biosynthesis in Vitro
A Direct Effect of Antiovulatory Compounds on Human Testicumr Biosynthesis in Vitro J. J.
BALATTI, M.D., A. CLARET, M.D., and
J.
M. ROSNER, M.D.
THE
REGULATION of reproductive capacity has been, in recent years, the goal of intense research effort. Large numbers of drugs with different chemical structures, grouped under the term "antiovulatory compounds," have been considered to inhibit the release of pituitary gonadotrophins.9 with the resulting blocking of ovulation in human females and spermatogenesis in human males. The discovery that ovulation can be inhibited in humans without an appreciable diminution of the urinary levels of gonadotrophins 1 • 4 • 5 • 7 • 10 • 11 has led some investigators to consider a probable direct effect of the antiovulatory compounds on the gonads. Previous work done in this laboratory shows that 17a-chloroethynyl-19-nor-4,9 ( 10) -androstadiene-17fi-ol-3-one was able to inhibit rat gonadal biosynthesis in vitro. 2 In a search for a direct effect on steroid biosynthesis, in the present experiment we have incubated human testes with labeled progesterone and 2 · different antiovulatories, 17cx-ethynyl-17fi-hydroxy-estr-4-ene ( lynestrenol), and 17a-chloroethynyl-19-nor-4,9 ( 10) -androstadiene-17j3-ol-3-one (MK 665).
MATERIAL AND METHODS
Human testes obtained from patients orchiectomized for prostatic carcinoma were prepared for homogenization by removing fat, tunica albuginea, epididymus, and epididymal fat pad. After being weighed, the tissue was cut in small pieces and homogenized in a Ten Broeck glass homogenizer* From the Laboratory of Endocrine Research, University del Salvador School of Medicine, the Department of Urology, Avellaneda Hospital, and the Consejo Nacional de Investigaciones Cientfficas y Tecnicas, Buenos Aires, Argentina. Supported by a grant from the Ford Foundation. The lynestrcnol and MK 665 used in this study were kindly supplied by· Ciba (Argentina) and Merck Sharp and Dohme, Rahway, N .•T., respectively. *Schaar Scientific Co., Chicago, III.
367
1
d
I
368
BALATTI ET AL.
FERTILITY
& STERILITY
in the cold. Sufficient 0.1 M phosphate buffer, pH 7.4, was added to make a 10% concentration of the homogenate. Progesterone-4-14C, Lot 79-238-36, * specific activity 70 pc.jmg., was used as substrate after being chromatographed on Whatman No. 1 papert in hexane-benzene-formamide. The equivalent of 625-780 mg. of tissue was used in each incubation flask. To each flask were added: adenosine triphosphate (ATP), 6.56 pmoles; nicotinamide adenine dinucleotide phosphate ( NADP), 3.60 pmoles; nicotinamide adenine dinucleotide ( NAD), 3.77 pmoles; glucose-6phosphate, 9.63 pmoles; and glucose-6-phosphate dehydrogenase, 50 pg. The final volume of 3.0 mi. was incubated for 2 hr. at 34 o C. under air in a Dubnoff metabolic shaker.t The incubation was stopped by the addition of 5 mi. of ether and extracted 3 times with 30 mi. of diethyl ether. The ether extract was washed twice with 10 mi. of 1 N NaOH and twice with 10 mi. of water, and was then filtered through sodium sulfate and dried under reduced pressure. The dried extract was applied to Whatman No. 1 paper and chromatographed in several Zaffaroni systems. 3 The first chromatography was performed in hexane-benzene-formamide for 3 hr. Previously, 100 pg. each of testosterone (17,8-hydroxy-~4 androsten-3-one), androstenedione ( ~ 4 -androsten-3,20-dione), and progesterone ( ~ 4 -pregnene-3,20-dione) was added to each flask as a reference standard and located, after being chromatographed, by the Haines scanner.§ The strips were scanned for radioactivity in an Actigraph Ill\ automatic strip scanner. Radioactivity was counted in a Tri-Carb liquid scintillation spectrometer Model 526, ~ using 10 mi. of scintillation fluid containing 4 gm. of 2,5-diphenyloxazole and 4 mg. of 1-4bis-2-( 4-methyl-5phenyloxazolyl) benzene-1-toluene. Counting efficiency for 14 C varied from 62 to 65%. The acetylation of the free compounds was done by adding 0.2 mi. of a mixture of acetic anhydride-pyridine ( 1.:5, v jv) to the dried extract. The acetylated sample was left overnight in the dark at room temperature ( 24280 C.), evaporated, and rechromatographed in hexane-formamide for 3 hr. The oxidation was performed with 0.2 mi. of 0.5% chromium trioxide in 95% glacial acetic acid for 2 hr. in the dark at room temperature; at the end of this time the reaction was stopped by adding 1 mi. of 20% ethanol. The extraction was done with 10 mi. of methylene dichloride and washed twice *New England Nuclear Corporation, Boston, Mass. tH. Reeve Angel Co., Clifton, N. J. :!:Precision Scientific Co., Chicago, Ill. §CAMAG, Muttenz, Switzerland. II Nuclear-Chicago Corp., Des Plaines, Ill. -,rPackard Instrument Co., Inc., Downers Grove, Ill.
VoL. 18, No. 3, 1961
369
ANTI<)vtiLAi'oii.Y Co:Mi>ouN'ns
with 1 ml. of water. Purity of the isolated steroids was ascertained for all the compounds by recrystallization to constant specific activity.
Experiment 1. A total of 22 flasks, each containing a 780-mg. aliquot from a pool of 4 human testes, was incubated after the addition of 1.45 p.g. of progesterone-4-14 C ( 2.2 X 105 disintegrations per minute [ dpm] ) as substrate to each flask. To 3 groups of 5 flasks each were added ( 1) 5 mg. of lynestrenol ( 1.7 X I0- 5 M); (2) 5 mg. of MK 665 (1.5 X IQ- 5 M); and (3) 2.5 mg. of MK 665 (7.5 X IQ- 6 M). To 7 flasks, no materials were added, the flasks being used as controls (Table 1). Experiment 2. A total of 40 flasks, each containing a 625-mg. aliquot from a pool of 6 human testes, was incubated after the addition of 1.45 p.g. of progesterone-4-14 C ( 2.2 X 105 dpm) as substrate to each flask. To 4 groups of 8 flasks each, MK 665 was added in the following amounts: (1) 1.25 mg. (3.75 X 10-oM); (2) 0.62 mg. (1.87 X IQ- 6 M); (3) 0.31 mg. (9.3 X I0- 7 M); and (4) 0.16 mg. (4.6 X IQ- 7 M). No TABLE 1. Production of Steroids with and Without the Addition of Antiovulatory Compounds from Progesterone-4-14 C ( 1.45 p.g., 2.2 X 105 dpm) by Human Testes in Vitro Steroid production (dpm) 20a-Hydrorey-4'pregnene-3-one
17a-Hydroreyprogesterone EXPERIME.II.'T
A ndrostenedione
Testosterone
1
Controls 4.2(±0.45)X104 3.3{±0.37)Xl04 1.1{±0.12)X104 8.4{±0.63)X103 MK 665 5 mg. Undet.* Undet. Undet. Undet. 2.5 mg. 8.0{±0.5) X103 1.5 ( ±0.31) X 103 Undet. Undet. Lynestrenol 5 mg. Undet. Undet. Undet. Undet. EXPERIMENT
Controls MK 665 1.25 mg. 0.62mg. 0.31 mg. 0.16 mg.
2
3.7 {±0.38) X104
2.1{±0.29)X104 3 ( ±0.41) X103
1.0 {±0.08) X 104 1.8{±0.15) X104 I.5{±0.10)X104 1.1 ( ±0.09) X104
2.8{ ±0.33) X103 1.9 {±0.20) X 103 2.1 {±0.27) X103 2.3 ( ±0.33) X104
Undet. Undet. Undet. Undet.
3.9(±0.37) X103 Undet. Undet. Undet. Undet.
Average tisme weight in each flask, Experiment I, was 780 mg.; in Experiment 2, 625 mg. *Undetectable production.
370
BALATTI ET AL.
FERTILITY
& STERILITY
MK 665 was added to a fifth group of 8 flasks which were used as controls (Table 1). RESULTS
Radioactive material having, on the first chromatogram, identical mobility with 17a.-hydroxyprogesterone (17 ex-hydroxy-~ 4 -pregnene-3,20-dione ), androstenedione, testosterone, 20a.-hydroxy-~4 -pregnene-3-one, and progesterone was processed as follows. Behavior Like That of 17a-Hydroxyprogesterone
This material did not form an acetate and had the same mobility in hexane-formamide ( 5 hr.) as authentic 17cx-hydroxyprogesterone before and after acetylation. When oxidized with chromium trioxide and rechromatographed for 3.5 hr. in hexane-benzene-formamide, and in toluenepropylene-glycol for 4.5 hr., the material behaved as carrier androstenedione. An aliquot of this material was recrystallized to constant specific activity from 3 different solvents after 20 mg. of authentic 17cx-hydroxyprogesterone was added (Table 2). Androstenedione-like Behavior
An aliquot of this material was acetylated and did not form a derivative. It had mobility similar to that of carrier androstenedione before and after acetylation when chromatographed for 3 hr. in hexane-formamide. It did
not oxidize either, and when rechromatographed in toluene-propyleneglycol for 4 hr. and in hexane-benzene-formamide for 3.5 hr., behaved as authentic androstenedione. Another aliquot was recrystallized to constant activity after 11 mg. of authentic androstenedione was added (Table 2). Testosterone-like Behavior
An aliquot of this material was acetylated and chromatographed on hexane-formamide for 3 hr. It had Rf* identical with authentic testosterone acetate that was chromatographed on 2 separate strips in the same system and localized by ultraviolet absorption. Another aliquot was chromatographed on hexane-formamide for 24 hr. in order to separate testosterone from 17cx-hydroxyprogesterone. It was then oxidized and rechromatographed on toluene-propylene-glycol for 2 hr. and had the same Rf as authentic androstenedione that was chromatographed on 2 separate strips *Rf
=
distance substance travels from the origin. distance solvent travels from the origin
VoL.
18,
No. 3,
1967
371
ANTIOVULATORY CoMPOUNDS
TABLE 2. Recrystallization of Material to Constant Specific Activity after Addition of Various Steroids to the Unknown Crystallization No.
Specific activity Solvent
17a·HYDROXYPROGESTERONE
1 2 3 4
(dpmfmg.) ADDED ( 20 MG.)
Ethanol:petroleum ether (8:2) Acetone Methanol:ethyl acetate (7:3) Ethanol:petroleum ether (8:2) ANDROSTENEDIONE ADDED (
1 2 3
ll
745 679 691 687
MG.)
Methanol Hexane: chloroform ( 3: 1) Ethanol
365 353 353
TESTOSTERONE ADDED ( 9 MG.)
I 2 3
Methanol Acetone Ethanol 20a-HYDROXY·A 4 -PREGNENE-3-0NE ADDED (
I 2 3
Ethyl acetate:hexane (7:3) Methanol:water (9:1) Ethanol
229 243
232
15
MG.)
935 96I
953
in the same system and localized by ultraviolet absorption. A final aliquot was recrystallized to constant specific activity after 9 mg. of authentic testosterone was added (Table 2). Behavior Like That of 20a-hydroxy-A 4 -pregnene-3-one
This material was acetylated and chromatographed in hexane-formamide for 4 hr. It had Rf identical with authentic 20~X-hydroxy-A4 -pregnene-3-one acetate that was chromatographed on 2 separate strips in the same system. The acetate was hydrolyzed with ethanolic potassium hydroxide, rechromatographed on toluene-propylene-glycol for 3 hr., and recrystallized to constant specific activity after 15 mg. of authentic 20~X-hydroxy-A 4 -preg nene-3-one was added (Table 2). Progesterone-like Behavior
On acetylation, this material did not form a derivative and had similar Rf to authentic progesterone when chromatographed for 3 hr. in hexane-
372
BALATTI ET AL.
FEUTILITY
& STEl\ILITY
formamide before and after acetylation. When oxidized with chromium trioxide and rechromatographed for 3 hr. in hexane-benzene-formamide and for 4 hr. in toluene-propylene-glycol, the material behaved as authentic progesterone. Progesterone-4- 14 C was converted to 17 a-hydroxyprogesterone, androstenedione, testosterone, and 20a-hydroxy-.l4 -pregnene-3-one by tissue from human testes obtained after castration for prostatic carcinoma. The production of steroids obtained by the controls may be seen in Table 1. The addition to the incubation flasks of 5 mg. ( 1.7 X 10- 5 M) of Lynestrenol produced a total inhibition of the testicular biosynthesis. When a similar amount of MK 665 ( 1.5 X 10- 5 M) was added to the incubation flasks, no utilization of the substrate was observed either. The addition of 2.5 mg. of MK 665 (7.5 X 10- 6 M) caused a marked diminution in the production of 20a-hydroxy-.l4 -pregnene-3-one and 17a-hydroxyprogesterone. Neither androstenedione nor testosterone was produced. The addition of smaller amounts of MK 665, ranging from 1.25 to 0.16 mg. ( 3.75 X 10- 6 M to 4.6 X 10- 7 M) caused, as shown in Table 1, a definite decrease in the synthesis of 20a-hydroxy-.l4 -pregnene-3-one and 1711-hydroxyprogesterone, except when the smallest dose was used, which inhibited the former steroid but not the latter. There was no production of tes~osterone or androstenedione detected from progesterone-4- 14 C. DISCUSSION
There have been many explanations of the mechanism of action of antiovulatory compounds, in an attempt to elucidate how they inhibit ovulation in females and spermatogenesis in males. The majority of investigators claim that these compounds act by inhibiting the release of pituitary gonadotrophins. Since clinical determinations of gonadotrophins do not discriminate between FSH and LH, it is impossible to establish whether the compounds selectively inhibit one or both gonadotrophins. Recent observations seem to indicate that some compounds suppress only LH and do not inhibit FSH. 14 Other drugs prevent ovulation by inhibiting FSH release from the pituitary gland, so there are no ripening follicles 6 able to respond to LHP On the other hand, Loraine has observed that some antiovulatory compounds are able to suppress ovulation without affecting the output of human pituitary gonadotrophins, and Lunenfeld et al. suggested that the antiovulatory capacity of 6a-methyl-17 a-acetoxyprogesterone, and 0.05 mg. of ethinyl estradiol may be due in part to a direct effect on the ovary,
VoL. I8, No. 3, I967
ANTiovuLATORY CoMPOUNDs
373
resulting in a decrease of its sensitivity to stimulation by pitmtary gonadotrophins. The foregoing observations are partially invalidated because in the majority of cases the antiovulatory compound was not the only drug utilized but was generally associated with estrogens. Our results show that 17a-chloroethynyl-19-nor-4,9 ( 10) -~-androstadiene17fi-ol-3-one, and 17a-ethynyl-17fi-hydroxy-estr-4-ene added in vitro to homogenates of human testes are able to inhibit the conversion of progesterone to 17a-hydroxyprogesterone, 20a-hydroxy-4-pregnene-3-one, and androgens by the testes. These results demonstrate that the 2 compounds employed in our experiments probably exert a direct effect on the activity of 2 human testicular enzymes related to the biosynthesis of androgens, and on the 20a-hydroxy steroid dehydrogenase. With the concentrations of both drugs employed, from IQ- 5 to I0- 7 M, no androgen biosynthesis was observed with the larger doses and only partial inhibition with the smaller. Although it is difficult to estimate the concentration of antiovulatory drugs that reaches a human gonad after oral ingestion or parenteral administration, I0- 7 M is rather a small amount. As Samuels et al. established when they demonstrated a direct effect of diethystilbestrol on testicular enzymes in Balb jC mice, the action could be either on the enzyme systems themselves or on the synthesis o~ the enzymes. Our results are only valid for our in-vitro experimental conditions, and it is not licit to extrapolate them to an in-vivo situation. At any rate, a direct effect on gonadal biosynthesis should not be overlooked when the mechanism of action of these compounds, either in experimental animals or in humans, is being considered. Laboratory of Endocrine Research University del Salvador School of Medicine Buenos Aires, Argentina
REFERENCES I. ALBERT, A., and SMITH, R. E. "Effect of Progesterone on HPG." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., I96I, p. 239. 2. BALATTI, J. J., CLARET, A., and RosNER, J. M. Unpublished data. 3. BunToN, R. B., ZAFFARONI, A., and KEUTMANN, E. H. Paper chromatography of steroids. II. Corticosteroids and related compounds. I Biol Chern 188:163, I951. 4. DicZFALUSY, E. "Urinary Gonadotropin Excretion with Previous Pituitary Irradiation." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., I96I, p. 257. 5. GREENBLATT, R. B., and MAHESH, V. B. Pituitary-ovarian relationships. Metabolism 14:320, I965. 6. LoRAINE, J. A. "The Effect of Contraceptive Drugs on Hormone Excretion During the Menstrual Cycle." In Pituitary-Ovarian Endocrinology (ed. I), Dorfman and Castro, Eds. Holden-Day, San Francisco, I963, p. 207.
374
BALATTI ET AL.
FERTILITY & STERILITY
7. LoRAINE, J., BRowN, J. B., and FoTHERBY, K. In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 222. 8. LUNENFELD, B., SULIMOVICI, S., and RABAU, E. Mechanism of action of antiovulatory compounds. ] Clin Endocr 23:391, 1963. 9. MARTIN, L., and CUNNINGHAM, K. "Inhibition of Human Pituitary Gonadotropin Output by 17-Ethinyl 19-nortestosterone." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 226. 10. McARTHUR, J. W., WoRCESTER, J., and INGERSOLL, F. M. "Effect of Single Dose of Progesterone Administered During Follicular Phase of Menstrual Cycle on Urinary ICSH Excretion and Cycle Length." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 231. II. MooRE, D. J., RoscoE, R. T., HELLER, C. G., and PAULSEN, C. A. "Failure of Progesterone to Depress Urinary Gonadotropin Excretion in Normal Menstruating Women." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 233. 12. OvERBEEK, G. A., and DE VISSER, J. Different modes of action of two antiovulatory compounds. Acta Endocr 45 (Supp. 90) :179, 1964. 13. SAMUELS, L. T., SHORT, J. G., and BusEBY, R. A. The effect of diethylstilboestrol on testicular 17-a-hydroxylase activities in Balb/C mice. Acta Endocr 45:487, 1964. 14. STEVENS, V. C., VoRYS, N., BEsH, P. K., and BARRY, R. D. The effects of a new oral contraceptive on gonadotropin excretion. Metabolism 14:327, 1965.
BALATTI, M.D., A. CLARET, M.D., and
J.
M. ROSNER, M.D.
THE
REGULATION of reproductive capacity has been, in recent years, the goal of intense research effort. Large numbers of drugs with different chemical structures, grouped under the term "antiovulatory compounds," have been considered to inhibit the release of pituitary gonadotrophins.9 with the resulting blocking of ovulation in human females and spermatogenesis in human males. The discovery that ovulation can be inhibited in humans without an appreciable diminution of the urinary levels of gonadotrophins 1 • 4 • 5 • 7 • 10 • 11 has led some investigators to consider a probable direct effect of the antiovulatory compounds on the gonads. Previous work done in this laboratory shows that 17a-chloroethynyl-19-nor-4,9 ( 10) -androstadiene-17fi-ol-3-one was able to inhibit rat gonadal biosynthesis in vitro. 2 In a search for a direct effect on steroid biosynthesis, in the present experiment we have incubated human testes with labeled progesterone and 2 · different antiovulatories, 17cx-ethynyl-17fi-hydroxy-estr-4-ene ( lynestrenol), and 17a-chloroethynyl-19-nor-4,9 ( 10) -androstadiene-17j3-ol-3-one (MK 665).
MATERIAL AND METHODS
Human testes obtained from patients orchiectomized for prostatic carcinoma were prepared for homogenization by removing fat, tunica albuginea, epididymus, and epididymal fat pad. After being weighed, the tissue was cut in small pieces and homogenized in a Ten Broeck glass homogenizer* From the Laboratory of Endocrine Research, University del Salvador School of Medicine, the Department of Urology, Avellaneda Hospital, and the Consejo Nacional de Investigaciones Cientfficas y Tecnicas, Buenos Aires, Argentina. Supported by a grant from the Ford Foundation. The lynestrcnol and MK 665 used in this study were kindly supplied by· Ciba (Argentina) and Merck Sharp and Dohme, Rahway, N .•T., respectively. *Schaar Scientific Co., Chicago, III.
367
1
d
I
368
BALATTI ET AL.
FERTILITY
& STERILITY
in the cold. Sufficient 0.1 M phosphate buffer, pH 7.4, was added to make a 10% concentration of the homogenate. Progesterone-4-14C, Lot 79-238-36, * specific activity 70 pc.jmg., was used as substrate after being chromatographed on Whatman No. 1 papert in hexane-benzene-formamide. The equivalent of 625-780 mg. of tissue was used in each incubation flask. To each flask were added: adenosine triphosphate (ATP), 6.56 pmoles; nicotinamide adenine dinucleotide phosphate ( NADP), 3.60 pmoles; nicotinamide adenine dinucleotide ( NAD), 3.77 pmoles; glucose-6phosphate, 9.63 pmoles; and glucose-6-phosphate dehydrogenase, 50 pg. The final volume of 3.0 mi. was incubated for 2 hr. at 34 o C. under air in a Dubnoff metabolic shaker.t The incubation was stopped by the addition of 5 mi. of ether and extracted 3 times with 30 mi. of diethyl ether. The ether extract was washed twice with 10 mi. of 1 N NaOH and twice with 10 mi. of water, and was then filtered through sodium sulfate and dried under reduced pressure. The dried extract was applied to Whatman No. 1 paper and chromatographed in several Zaffaroni systems. 3 The first chromatography was performed in hexane-benzene-formamide for 3 hr. Previously, 100 pg. each of testosterone (17,8-hydroxy-~4 androsten-3-one), androstenedione ( ~ 4 -androsten-3,20-dione), and progesterone ( ~ 4 -pregnene-3,20-dione) was added to each flask as a reference standard and located, after being chromatographed, by the Haines scanner.§ The strips were scanned for radioactivity in an Actigraph Ill\ automatic strip scanner. Radioactivity was counted in a Tri-Carb liquid scintillation spectrometer Model 526, ~ using 10 mi. of scintillation fluid containing 4 gm. of 2,5-diphenyloxazole and 4 mg. of 1-4bis-2-( 4-methyl-5phenyloxazolyl) benzene-1-toluene. Counting efficiency for 14 C varied from 62 to 65%. The acetylation of the free compounds was done by adding 0.2 mi. of a mixture of acetic anhydride-pyridine ( 1.:5, v jv) to the dried extract. The acetylated sample was left overnight in the dark at room temperature ( 24280 C.), evaporated, and rechromatographed in hexane-formamide for 3 hr. The oxidation was performed with 0.2 mi. of 0.5% chromium trioxide in 95% glacial acetic acid for 2 hr. in the dark at room temperature; at the end of this time the reaction was stopped by adding 1 mi. of 20% ethanol. The extraction was done with 10 mi. of methylene dichloride and washed twice *New England Nuclear Corporation, Boston, Mass. tH. Reeve Angel Co., Clifton, N. J. :!:Precision Scientific Co., Chicago, Ill. §CAMAG, Muttenz, Switzerland. II Nuclear-Chicago Corp., Des Plaines, Ill. -,rPackard Instrument Co., Inc., Downers Grove, Ill.
VoL. 18, No. 3, 1961
369
ANTI<)vtiLAi'oii.Y Co:Mi>ouN'ns
with 1 ml. of water. Purity of the isolated steroids was ascertained for all the compounds by recrystallization to constant specific activity.
Experiment 1. A total of 22 flasks, each containing a 780-mg. aliquot from a pool of 4 human testes, was incubated after the addition of 1.45 p.g. of progesterone-4-14 C ( 2.2 X 105 disintegrations per minute [ dpm] ) as substrate to each flask. To 3 groups of 5 flasks each were added ( 1) 5 mg. of lynestrenol ( 1.7 X I0- 5 M); (2) 5 mg. of MK 665 (1.5 X IQ- 5 M); and (3) 2.5 mg. of MK 665 (7.5 X IQ- 6 M). To 7 flasks, no materials were added, the flasks being used as controls (Table 1). Experiment 2. A total of 40 flasks, each containing a 625-mg. aliquot from a pool of 6 human testes, was incubated after the addition of 1.45 p.g. of progesterone-4-14 C ( 2.2 X 105 dpm) as substrate to each flask. To 4 groups of 8 flasks each, MK 665 was added in the following amounts: (1) 1.25 mg. (3.75 X 10-oM); (2) 0.62 mg. (1.87 X IQ- 6 M); (3) 0.31 mg. (9.3 X I0- 7 M); and (4) 0.16 mg. (4.6 X IQ- 7 M). No TABLE 1. Production of Steroids with and Without the Addition of Antiovulatory Compounds from Progesterone-4-14 C ( 1.45 p.g., 2.2 X 105 dpm) by Human Testes in Vitro Steroid production (dpm) 20a-Hydrorey-4'pregnene-3-one
17a-Hydroreyprogesterone EXPERIME.II.'T
A ndrostenedione
Testosterone
1
Controls 4.2(±0.45)X104 3.3{±0.37)Xl04 1.1{±0.12)X104 8.4{±0.63)X103 MK 665 5 mg. Undet.* Undet. Undet. Undet. 2.5 mg. 8.0{±0.5) X103 1.5 ( ±0.31) X 103 Undet. Undet. Lynestrenol 5 mg. Undet. Undet. Undet. Undet. EXPERIMENT
Controls MK 665 1.25 mg. 0.62mg. 0.31 mg. 0.16 mg.
2
3.7 {±0.38) X104
2.1{±0.29)X104 3 ( ±0.41) X103
1.0 {±0.08) X 104 1.8{±0.15) X104 I.5{±0.10)X104 1.1 ( ±0.09) X104
2.8{ ±0.33) X103 1.9 {±0.20) X 103 2.1 {±0.27) X103 2.3 ( ±0.33) X104
Undet. Undet. Undet. Undet.
3.9(±0.37) X103 Undet. Undet. Undet. Undet.
Average tisme weight in each flask, Experiment I, was 780 mg.; in Experiment 2, 625 mg. *Undetectable production.
370
BALATTI ET AL.
FERTILITY
& STERILITY
MK 665 was added to a fifth group of 8 flasks which were used as controls (Table 1). RESULTS
Radioactive material having, on the first chromatogram, identical mobility with 17a.-hydroxyprogesterone (17 ex-hydroxy-~ 4 -pregnene-3,20-dione ), androstenedione, testosterone, 20a.-hydroxy-~4 -pregnene-3-one, and progesterone was processed as follows. Behavior Like That of 17a-Hydroxyprogesterone
This material did not form an acetate and had the same mobility in hexane-formamide ( 5 hr.) as authentic 17cx-hydroxyprogesterone before and after acetylation. When oxidized with chromium trioxide and rechromatographed for 3.5 hr. in hexane-benzene-formamide, and in toluenepropylene-glycol for 4.5 hr., the material behaved as carrier androstenedione. An aliquot of this material was recrystallized to constant specific activity from 3 different solvents after 20 mg. of authentic 17cx-hydroxyprogesterone was added (Table 2). Androstenedione-like Behavior
An aliquot of this material was acetylated and did not form a derivative. It had mobility similar to that of carrier androstenedione before and after acetylation when chromatographed for 3 hr. in hexane-formamide. It did
not oxidize either, and when rechromatographed in toluene-propyleneglycol for 4 hr. and in hexane-benzene-formamide for 3.5 hr., behaved as authentic androstenedione. Another aliquot was recrystallized to constant activity after 11 mg. of authentic androstenedione was added (Table 2). Testosterone-like Behavior
An aliquot of this material was acetylated and chromatographed on hexane-formamide for 3 hr. It had Rf* identical with authentic testosterone acetate that was chromatographed on 2 separate strips in the same system and localized by ultraviolet absorption. Another aliquot was chromatographed on hexane-formamide for 24 hr. in order to separate testosterone from 17cx-hydroxyprogesterone. It was then oxidized and rechromatographed on toluene-propylene-glycol for 2 hr. and had the same Rf as authentic androstenedione that was chromatographed on 2 separate strips *Rf
=
distance substance travels from the origin. distance solvent travels from the origin
VoL.
18,
No. 3,
1967
371
ANTIOVULATORY CoMPOUNDS
TABLE 2. Recrystallization of Material to Constant Specific Activity after Addition of Various Steroids to the Unknown Crystallization No.
Specific activity Solvent
17a·HYDROXYPROGESTERONE
1 2 3 4
(dpmfmg.) ADDED ( 20 MG.)
Ethanol:petroleum ether (8:2) Acetone Methanol:ethyl acetate (7:3) Ethanol:petroleum ether (8:2) ANDROSTENEDIONE ADDED (
1 2 3
ll
745 679 691 687
MG.)
Methanol Hexane: chloroform ( 3: 1) Ethanol
365 353 353
TESTOSTERONE ADDED ( 9 MG.)
I 2 3
Methanol Acetone Ethanol 20a-HYDROXY·A 4 -PREGNENE-3-0NE ADDED (
I 2 3
Ethyl acetate:hexane (7:3) Methanol:water (9:1) Ethanol
229 243
232
15
MG.)
935 96I
953
in the same system and localized by ultraviolet absorption. A final aliquot was recrystallized to constant specific activity after 9 mg. of authentic testosterone was added (Table 2). Behavior Like That of 20a-hydroxy-A 4 -pregnene-3-one
This material was acetylated and chromatographed in hexane-formamide for 4 hr. It had Rf identical with authentic 20~X-hydroxy-A4 -pregnene-3-one acetate that was chromatographed on 2 separate strips in the same system. The acetate was hydrolyzed with ethanolic potassium hydroxide, rechromatographed on toluene-propylene-glycol for 3 hr., and recrystallized to constant specific activity after 15 mg. of authentic 20~X-hydroxy-A 4 -preg nene-3-one was added (Table 2). Progesterone-like Behavior
On acetylation, this material did not form a derivative and had similar Rf to authentic progesterone when chromatographed for 3 hr. in hexane-
372
BALATTI ET AL.
FEUTILITY
& STEl\ILITY
formamide before and after acetylation. When oxidized with chromium trioxide and rechromatographed for 3 hr. in hexane-benzene-formamide and for 4 hr. in toluene-propylene-glycol, the material behaved as authentic progesterone. Progesterone-4- 14 C was converted to 17 a-hydroxyprogesterone, androstenedione, testosterone, and 20a-hydroxy-.l4 -pregnene-3-one by tissue from human testes obtained after castration for prostatic carcinoma. The production of steroids obtained by the controls may be seen in Table 1. The addition to the incubation flasks of 5 mg. ( 1.7 X 10- 5 M) of Lynestrenol produced a total inhibition of the testicular biosynthesis. When a similar amount of MK 665 ( 1.5 X 10- 5 M) was added to the incubation flasks, no utilization of the substrate was observed either. The addition of 2.5 mg. of MK 665 (7.5 X 10- 6 M) caused a marked diminution in the production of 20a-hydroxy-.l4 -pregnene-3-one and 17a-hydroxyprogesterone. Neither androstenedione nor testosterone was produced. The addition of smaller amounts of MK 665, ranging from 1.25 to 0.16 mg. ( 3.75 X 10- 6 M to 4.6 X 10- 7 M) caused, as shown in Table 1, a definite decrease in the synthesis of 20a-hydroxy-.l4 -pregnene-3-one and 1711-hydroxyprogesterone, except when the smallest dose was used, which inhibited the former steroid but not the latter. There was no production of tes~osterone or androstenedione detected from progesterone-4- 14 C. DISCUSSION
There have been many explanations of the mechanism of action of antiovulatory compounds, in an attempt to elucidate how they inhibit ovulation in females and spermatogenesis in males. The majority of investigators claim that these compounds act by inhibiting the release of pituitary gonadotrophins. Since clinical determinations of gonadotrophins do not discriminate between FSH and LH, it is impossible to establish whether the compounds selectively inhibit one or both gonadotrophins. Recent observations seem to indicate that some compounds suppress only LH and do not inhibit FSH. 14 Other drugs prevent ovulation by inhibiting FSH release from the pituitary gland, so there are no ripening follicles 6 able to respond to LHP On the other hand, Loraine has observed that some antiovulatory compounds are able to suppress ovulation without affecting the output of human pituitary gonadotrophins, and Lunenfeld et al. suggested that the antiovulatory capacity of 6a-methyl-17 a-acetoxyprogesterone, and 0.05 mg. of ethinyl estradiol may be due in part to a direct effect on the ovary,
VoL. I8, No. 3, I967
ANTiovuLATORY CoMPOUNDs
373
resulting in a decrease of its sensitivity to stimulation by pitmtary gonadotrophins. The foregoing observations are partially invalidated because in the majority of cases the antiovulatory compound was not the only drug utilized but was generally associated with estrogens. Our results show that 17a-chloroethynyl-19-nor-4,9 ( 10) -~-androstadiene17fi-ol-3-one, and 17a-ethynyl-17fi-hydroxy-estr-4-ene added in vitro to homogenates of human testes are able to inhibit the conversion of progesterone to 17a-hydroxyprogesterone, 20a-hydroxy-4-pregnene-3-one, and androgens by the testes. These results demonstrate that the 2 compounds employed in our experiments probably exert a direct effect on the activity of 2 human testicular enzymes related to the biosynthesis of androgens, and on the 20a-hydroxy steroid dehydrogenase. With the concentrations of both drugs employed, from IQ- 5 to I0- 7 M, no androgen biosynthesis was observed with the larger doses and only partial inhibition with the smaller. Although it is difficult to estimate the concentration of antiovulatory drugs that reaches a human gonad after oral ingestion or parenteral administration, I0- 7 M is rather a small amount. As Samuels et al. established when they demonstrated a direct effect of diethystilbestrol on testicular enzymes in Balb jC mice, the action could be either on the enzyme systems themselves or on the synthesis o~ the enzymes. Our results are only valid for our in-vitro experimental conditions, and it is not licit to extrapolate them to an in-vivo situation. At any rate, a direct effect on gonadal biosynthesis should not be overlooked when the mechanism of action of these compounds, either in experimental animals or in humans, is being considered. Laboratory of Endocrine Research University del Salvador School of Medicine Buenos Aires, Argentina
REFERENCES I. ALBERT, A., and SMITH, R. E. "Effect of Progesterone on HPG." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., I96I, p. 239. 2. BALATTI, J. J., CLARET, A., and RosNER, J. M. Unpublished data. 3. BunToN, R. B., ZAFFARONI, A., and KEUTMANN, E. H. Paper chromatography of steroids. II. Corticosteroids and related compounds. I Biol Chern 188:163, I951. 4. DicZFALUSY, E. "Urinary Gonadotropin Excretion with Previous Pituitary Irradiation." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., I96I, p. 257. 5. GREENBLATT, R. B., and MAHESH, V. B. Pituitary-ovarian relationships. Metabolism 14:320, I965. 6. LoRAINE, J. A. "The Effect of Contraceptive Drugs on Hormone Excretion During the Menstrual Cycle." In Pituitary-Ovarian Endocrinology (ed. I), Dorfman and Castro, Eds. Holden-Day, San Francisco, I963, p. 207.
374
BALATTI ET AL.
FERTILITY & STERILITY
7. LoRAINE, J., BRowN, J. B., and FoTHERBY, K. In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 222. 8. LUNENFELD, B., SULIMOVICI, S., and RABAU, E. Mechanism of action of antiovulatory compounds. ] Clin Endocr 23:391, 1963. 9. MARTIN, L., and CUNNINGHAM, K. "Inhibition of Human Pituitary Gonadotropin Output by 17-Ethinyl 19-nortestosterone." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 226. 10. McARTHUR, J. W., WoRCESTER, J., and INGERSOLL, F. M. "Effect of Single Dose of Progesterone Administered During Follicular Phase of Menstrual Cycle on Urinary ICSH Excretion and Cycle Length." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 231. II. MooRE, D. J., RoscoE, R. T., HELLER, C. G., and PAULSEN, C. A. "Failure of Progesterone to Depress Urinary Gonadotropin Excretion in Normal Menstruating Women." In Human Pituitary Gonadotropins. Thomas, Springfield, Ill., 1961, p. 233. 12. OvERBEEK, G. A., and DE VISSER, J. Different modes of action of two antiovulatory compounds. Acta Endocr 45 (Supp. 90) :179, 1964. 13. SAMUELS, L. T., SHORT, J. G., and BusEBY, R. A. The effect of diethylstilboestrol on testicular 17-a-hydroxylase activities in Balb/C mice. Acta Endocr 45:487, 1964. 14. STEVENS, V. C., VoRYS, N., BEsH, P. K., and BARRY, R. D. The effects of a new oral contraceptive on gonadotropin excretion. Metabolism 14:327, 1965.