The roles of estrogen and progestin in producing deciduosarcoma and other lesions in the rabbit.

The interactions of estrogens and progestins in producing decidualization, deciduosarcoma. and other lesions in the rabbit were explored. Steroids were delivered by silicone elastomer implants placed subdermally except for oral dosing in 1 experiment. Varying doses of levonorgestrel (LNG) were given with and without estradiol (E2) and varying doses of E2 with and without LNG. LNG alone delivered at an estimated mean dose of 233 microg/day did not result in endometrial decidualization or deciduosarcoma. Both conditions occurred when E2 was added to the regimen and increased as the dose of E2 was increased. Sixty microg of E2 per day produced endometrial decidualization in all test animals in a 2-month exposure, but deciduosarcoma occurred only when LNG was also supplied and increased as the LNG dose was increased. Progesterone given with E2 resulted in deciduosarcoma in most rabbits. Ethynylestradiol alone at 30 microg/day delivered by implants produced splenic and ovarian deciduosarcomas in 1 of 5 test animals. Adding LNG resulted in more numerous and widespread deciduosarcomas. These experiments indicate that exogenous estrogen is necessary for decidualization of the endometrium and to production of deciduosarcoma in the nonpregnant rabbit. Exogenous progestin promotes the process. Necrosis of the uterine wall tended to increase with increasing dose of estrogens.

The biological activity of 7 alpha-methyl-19-nortestosterone is not amplified in male reproductive tract as is that of testosterone.

Based on the premise that testosterone, but not 7 alpha-methyl-androgens, is reduced at the 5 alpha-position in the prostate and seminal vesicles, the differential bioactivities of these androgens were investigated in castrated rats. The ability of 7 alpha-methyl-19-nortestosterone acetate (MENT) to increase the weights of ventral prostate and seminal vesicles of castrated rats was four times higher than that of testosterone, while its effect on the weights of bulbocavernosus plus levator ani muscles (muscle), was 10 times that of testosterone. MENT was also approximately 12 times more potent than testosterone in the suppression of serum gonadotropin levels. A dose of testosterone that maintains serum gonadotropin levels and muscle mass also maintains prostate and seminal vesicle weights in castrated rats. By contrast, a dose of MENT that maintains muscle and gonadotropins does not maintain prostate and seminal vesicles. The action of other 7 alpha-methylated androgens were similar to that of MENT. The importance of 5 alpha reductase in the differential action of testosterone and MENT on prostate was confirmed by using a 5 alpha-reductase inhibitor. The activity of testosterone was significantly suppressed in the ventral prostate and seminal vesicles but not on muscle by the 5 alpha-reductase inhibitor (N,N-diethyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide). The enzyme inhibitor, however, had no influence on the activity of MENT on either tissue. In contrast, cyproterone acetate, an antiandrogen that competitively binds to the androgen receptors, inhibited the action of MENT and of testosterone on the prostate as well as on the muscle. In conclusion, these observations show that 7 alpha-methylated androgens can maintain muscle mass and normal gonadotropin levels in androgen deficient rats without hyperstimulating the prostate. These findings suggest that 7 alpha-methylated androgens may offer some health benefits to men who require androgen treatment.

Radioimmunoassay of 7 alpha-methyl-19-nortestosterone and investigation of its pharmacokinetics in animals.

A method for the measurement of 7 alpha-methyl-19-nortestosterone (7MENT) in serum/plasma by radioimmunoassay (RIA) is described. The antiserum, raised against 7 alpha-methyl-19-nortestosterone-3-O-oxime-bovine serum albumin, had a low titer (final dilution = 1:4500) and low affinity (Ka = 1.17 x 10(9) l/mol) but showed little or no cross-reactivity with several of the steroids tested. The sensitivity of the RIA was 28.2 pg/ml and the mean recovery of added cold steroid was 86 to 100%. Intra- and inter-assay coefficients of variation ranged from 4.3 to 7.3% and 7.3 to 8.4%, respectively. This RIA was used to follow plasma 7MENT levels after a single i.v. injection of the steroid in rats and rabbits. The metabolic clearance rates (MCR) of 7MENT as determined from the plasma disappearance curve for rats and rabbits were 50 l/day and 336 l/day, respectively. The MCR of 7MENT in rats and rabbits lies in the same range as for testosterone. When compared to other nortestosterone derivatives such as norethisterone, 7MENT is metabolized relatively faster.

A 90-day subcutaneous toxicity and fertility study of a LHRH antagonist in rats.

[Ac-D2Nal1,4Cl-DPhe2,D3Pal3,Arg5,DGlu6+ ++ (anisole adduct),DAla10]-GnRH (Nal-Glu) is an antagonist of LHRH and has the potential to be utilized as an antigonadal agent. A study was undertaken to evaluate the toxicological effects of Nal-Glu in rats. Nal-Glu, dissolved in 5% mannitol in water containing 9 ml/liter benzyl alcohol, was administered subcutaneously. In subchronic studies, groups of 12 male and 12 female rats received 0, 50, 250, or 1250 micrograms/kg body weight (BW) Nal-Glu for 90 days and were killed on Day 91. Additional groups of male and female rats were given the high dose of Nal-Glu (1250 micrograms/kg BW) or vehicle for either 30 or 90 days. Their fertility was assessed by mating them with normal animals. Unlike some other LHRH antagonists, Nal-Glu exhibited a low potency for causing in vitro histamine release from rat peritoneal mast cells. Furthermore, in acute in vivo studies, Nal-Glu was less active in the induction of peripheral edema. In the subchronic study, all doses of Nal-Glu were well tolerated and there were no apparent systemic toxic effects. The pharmacological effects of Nal-Glu were quite evident, however. Nal-Glu treatment led to a significantly decreased body weight gain in the males and a significantly increased body weight gain in the females. There was a dose-dependent decrease in weights of gonads and reproductive organs in both the sexes. Some of the hematological and serological parameters were significantly different in Nal-Glu-treated animals. However, most of the values were within the normal range and are considered to be of no toxicological significance. Histopathological evaluations were made in the control and high-dose groups only. In the male, a seminiferous tubular degeneration and atrophy of the interstitial cells was seen. The prostate and seminal vesicles were also atrophied and the epididymides were devoid of spermatozoa. In the females, the ovaries and uteri were atrophic. The injection site of Nal-Glu-treated rats had inflammatory changes indicative of a local irritating action of the drug. All other tissues had normal histomorphology. Both male and female rats became infertile when 1250 micrograms/kg Nal-Glu was administered for 30 days. Normal fertility was restored 8 weeks after cessation of 90-day treatment. It is concluded that repeated administration of Nal-Glu leads to reversible infertility in both male and female rats. Although it was irritating at the site of injection. Nal-Glu had no systemic toxicological effects.

Mechanism of LHRH-stimulated steroidogenesis in rat Leydig cells: lipoxygenase products of arachidonic acid may not be involved.

Luteinizing hormone releasing hormone agonist, [(imBzl)-DHis6,Pro9,NEt]-LHRH (LHRH-A), caused a two to threefold increase in in vitro testosterone (T) secretion by rat Leydig cells. This LHRH-A-induced T secretion was completely blocked by quinacrine and chloroquine, inhibitors of phospholipase A2. Addition of phospholipase A2, however, was ineffective in stimulating basal or LHRH-A-induced T secretion. Phospholipase C, on the other hand, significantly stimulated both basal and LHRH-A-induced T secretion. Exogenously added arachidonic acid stimulated basal T secretion in a dose dependent manner, the maximum increase being about 100% over basal at a dose of 100 microM. Higher doses of arachidonic acid had no stimulatory effect. In the presence of LHRH-A, the stimulatory effect of arachidonic acid was additive up to a concentration of 100 microM; but higher concentrations of arachidonic acid (200 microM) were inhibitory. LHRH-A-induced steroidogenesis was inhibited by 5, 8, 11, 14 Eicosatetraynoic acid (ETYA), an inhibitor of all the three known pathways of arachidonic acid metabolism, and by nordihydroguaiaretic acid, and inhibitory of the lipoxygenase pathway of arachidonic acid metabolism. LHRH-A-stimulated T secretion was not inhibited by indomethacin, an inhibitor of the cyclo-oxygenase pathway of arachidonic acid metabolism. ETYA inhibited arachidonic acid-induced T secretion. Nordihydroguaiaretic acid, on the other hand, augmented basal, arachidonic acid-, phospholipase C-, or phorbol 12, myristate 13 acetate-induced testosterone secretion. These results suggest that arachidonic acid, whose release is influenced by phospholipase C, is involved in LHRH-A-induced T secretion by rat Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sulfasalazine and its analogs on fertility in male rats.

Several derivatives of sulfasalazine were tested for their antifertility activity in male rats. The compounds were administered to groups of rats daily by oral gavage for 28 days. Fertility of the rats treated with sulfasalazine or compound CH 74A was reduced, while other compounds had no effect. In a subsequent experiment, therefore, only the active compounds were studied further. Fertility of rats treated with sulfasalazine, compound CH 74A, CH 99A or sulfapyridine was reduced during 40 days of treatment. At the end of treatment, body weights were reduced in higher dose groups of sulfasalazine, CH 74A and sulfapyridine compared to control animals. The weights of the testes, prostate or seminal vesicle were not altered by any of the treatments. On the other hand, weight of the epididymides decreased in all higher dose groups except in CH 99A-treated animals. Sperm motility decreased in all the treated rats except in animals treated with low dose of sulfapyridine, whereas epididymal sperm count decreased in all but CH 99A-treated animals. These results suggest that sulfasalazine and its derivatives bring about their antifertility effects by decreasing sperm motility and/or number of spermatozoa.

Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells.

Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to inositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA), a tumor promoting agent, could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA (100 mU and 12.5 microM, respectively) augmented hCG induced T secretion, while at higher concentrations (PLC: 500 mU and AA: 200 microM) they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5, 8, 11, 14 Eicosatetraynoic acid (ETYA), a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. We conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclooxygenase products.

Comparison of the antispermatogenic effects of a new D-homo-steroid and testosterone in rabbits.

In comparison to testosterone, 18 beta-hydroxy-18 alpha-methyl-16 alpha-methylene-D-homo-5 alpha-androstane-3-one (D-homo-S) shows more pronounced anti-gonadotrophic than androgenic properties in rats. The present study was initiated in rabbits to investigate the potential of D-homo-S to suppress spermatogenesis. D-homo-S in sesame oil was administered at the doses of 0.1 (DI), 3 (D II) or 10 mg (D III) per rabbit each day for 8 weeks. During treatment serum testosterone, sperm concentration and quality of sperm motility decreased, whereas sex drive, semen volume and seminal plasma concentrations of fructose and zinc were not changed in any of the groups. Testicular weight and intratesticular testosterone concentration decreased significantly in groups D II and D III, while weights of accessory sex glands increased in those groups. Testosterone in the same dose regimen did not suppress sperm count, motility or serum testosterone, however, seminal plasma zinc concentration in group T III and fructose in group TI increased. Testicular weight and intratesticular testosterone concentration decreased in group TIII only. On the other hand, the weight of the accessory sex glands increased in the same group. In conclusion, D-homo-S suppresses spermatogenesis and increases accessory sex gland weights at doses, when testosterone is still ineffective. Thus, in rabbits D-homo-S appears to be a more potent androgen than testosterone but a dissociation between antigonadotrophic and androgenic properties could not be observed.

Effect of prostaglandins A-1, E-2 and F-2 alpha on blood plasma levels of testosterone, LH and FSH in male rats.

Effects of the administration of PGs A-1, E-2 and F-2 alpha (150 micrograms/rat b.i.d. for 10 days) were studied. Significant increase in testicular weight was observed only in PGE-2 treated group. Testicular ascorbic acid content reduced significantly by treatment with all the PGs. PGE-2 treatment caused a significant decrease in the content of testicular cholesterol, while no change was observed in the same and prostatic acid phosphatase activity in any of the PG treated groups. Blood plasma levels of testosterone drastically reduced by both PGE-2 and PGF-2 alpha, while there was no change in the levels of plasma LH in any of the groups. Plasma FSH levels increased significantly in PGA-1 treated rats only. The results suggest that 1) There is a direct action of PG particularly PGE-2 on testicular weight. PGE-2 increases testicular weight possibly by preventing degeneration of spermatids, 2) PGE-2 acting directly on the testis, reduces testicular ascorbic acid content, stimulates the conversion of cholesterol to pregnenolone but depresses the conversion of the latter to testosterone.

Effect of aspirin on spermatogenesis in mature and immature rats.

Aspirin administration for 30 days (5 mg/100 g body weight) caused a significant decrease in the weight of testis of immature rats. Decrease in the activities of sorbitol dehydrogenase and hyaluronidase was observed in both immature and mature rats. Decrease in the number of spermatids and increase in size of spermatocytes nuclei were observed. It is concluded that aspirin causes impairment of the later stages of spermatogenesis.

Effects of aspirin on blood plasma levels of testosterone, LH and FSH in maturing male rats.

Effects of aspirin administration (5 mg/100 g body weight) for various days were observed on the hormonal levels of maturing male rats. Aspirin was given orally every day for 8, 15, 22 and 30 days. No change in the weights of testes, epididymis, prostate or seminal vesicles was observed after treatment. While aspirin administration for 8 days caused an increase in the plasma testosteone level with decrease in both LH and FSH levels, prolonged treatment for 15 days and more produced a reverse effect, viz. decrease in plasma testosterone and increase in plasma LH and FSH levels. Testicular ascorbic acid content was found to decrease on the 15th and the 22nd day of treatment. Testicular cholesterol was increased after 22nd and 30th days of treatment. Prostatic acid phosphatase activity decreased in all the treated groups. The possible significance of these findings is discussed.

Effects of prostaglandins E-1, E-2, F-1a and F-2a on human sperm motility.

Effects of the addition of 100 micrograms each of prostaglandins E1, E2, F1a and F2a were seen on the motility, fructose utilization and total ATPase activity of human spermatozoa. PGE2 enhanced sperm motility and fructose utilization and decreased the whole sperm ATPase activity. While PGE1 had no effect on any of the parameters studied, PGF1a and PGF2a decreased sperm motility to some extent. Fructose utilization was reduced after PGF1a addition only. No change in the activity of ATPase was observed after treatment with F series of prostaglandins.

Effect of prostaglandins A-1, E-2 and F-2 alpha on spermatogenesis in rats.

Rats were treated with twice daily injections of 100 microgram PG/rat for 15 days. PGA-1 had no effect. PGE-2 caused a significant increase in testicular weight, RNA content, hyaluronidase activity and number of spermatids. PGF-2 alpha produced a significant decrease in sorbitol dehydrogenase activity and DNA content. It is suggested that PGE-2 may be involved in later stages of spermatogenesis, i.e. conversion of spermatocytes to spermatids.