The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study 45-d-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIAs or ELISAs, and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-β1, N-cadherin, α-catenin, and β-catenin protein at 24 h and later time points. Increase in expression of Fas ligand and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022-treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of stem cell factor, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.
The present study was undertaken to examine the antispermatogenic effect of l-CDB-4022 in the adult male cynomolgus monkey. Monkeys (four per group) were dosed via nasogastric tube for 7 d with l-CDB-4022 at 12.5 mg/kg.d or vehicle (d 0=first day of dosing). Plasma levels of l-CDB-4022 and its deesterified metabolite were nondetectable prior to treatment and in all vehicle-treated monkeys. Peak levels of l-CDB-4022 and its metabolite were observed at 4 h after dosing with steady-state levels apparent around d 4. Sperm concentration and total sperm per ejaculate were decreased to levels below 1x10(6) sperm/ml or sperm/ejaculate in l-CDB-4022-treated monkeys by d 17 and remained suppressed through wk 6. Sperm motility also declined to 0% for 6 wk. Testicular volume was reduced in l-CDB-4022-treated monkeys through d 21. The left testis and epididymis were removed from all monkeys on d 24. At this time, the most mature germ cells in the seminiferous tubules of testes from l-CDB-4022-treated monkeys were either spermatocytes or round spermatids. Immature germ cells, but not mature sperm, were found in the efferent ducts and collapsed epididymal lumen of l-CDB-4022-treated monkeys. A steady recovery in sperm motility, concentration, and total sperm per ejaculate was observed in l-CDB-4022-treated monkeys such that these parameters were not different from those of vehicle-treated monkeys by wk 16. Volume of the remaining testis increased in vehicle- and l-CDB-4022-treated monkeys after hemicastration; however, the increase in l-CDB-4022-treated monkeys was delayed compared with that observed in the vehicle-treated monkeys. The morphology of the remaining testis and epididymis, which were removed on wk 17, was normal. Serum inhibin B levels were increased in l-CDB-4022-treated monkeys during the dosing interval; thereafter serum inhibin B levels declined such that there was no difference between the groups by wk 3. l-CDB-4022 treatment did not affect circulating levels of testosterone, LH, FSH, or estradiol. In conclusion, these data indicate that in the cynomolgus monkey, a representative higher primate, l-CDB-4022 exerts a selective antispermatogenic action, which was reversible under the conditions of this study and thus has potential as a nonhormonal oral male contraceptive.
The potent androgens dimethandrolone 17β-undecanoate (DMAU) and 11β-methyl-19-nortestosterone 17β-dodecylcarbonate (11β-MNTDC) are in development for androgen replacement therapy and hormonal contraception in men. They can be delivered either orally or as long-acting injectables. In the current study, their long-term effects on body composition (percentage lean and fat mass); bone mineral density (BMD); serum gonadotropin levels; and weights of the prostate, seminal vesicles, and levator ani muscle were assessed. Four-week-old male rats were sham-operated (intact) or castrated (Cx) and treated subcutaneously for 16 weeks postsurgery with vehicle (Cx, intact), DMAU, or 11β-MNTDC every 4 weeks; testosterone enanthate (TE) every 2 weeks; or a testosterone (T) implant. There were significant differences in body weights over time with a general trend of intact = Cx + T = Cx + TE > Cx + 11β-MNTDC > Cx > Cx + DMAU. At week 18, rats were evaluated by dual-energy x-ray absorptiometry using the whole-body function of the Hologic software. The percentage lean body mass and BMD were lower (P < .05) in Cx rats than intact rats but equivalent in all groups of androgen-treated Cx rats and intact rats (P > .05). The highest percentage body fat was observed in Cx rats. Only DMAU- and 11β-MNTDC-treated rats had lower percentage body fat compared with Cx rats (P < .05). Prostate, seminal vesicles, and levator ani muscle weights, corrected for final body weight, were decreased (P < .05) in Cx compared with intact rats and increased to varying extents in androgen-treated Cx rats compared with Cx rats (P < .05). The most marked increases were observed in the DMAU-treated rats in which prostate and seminal vesicle weights/kg body weight were 2.4 to 2.7 times those of intact rats, and levator ani muscle weights were increased approximately 1.5-fold. Blood was collected from the tail vein at weeks 4, 8, 12, and 16 for measurement of serum levels of androgens and at necropsy at week 18 for measurement of serum gonadotropins. Serum levels of luteinizing hormone and follicle-stimulating hormone were greatly elevated in Cx rats at week 18 and suppressed to levels comparable to those in intact rats by DMAU, 11β-MNTDC, and T implants (P > .05). Collectively, our data indicate that androgen replacement with DMAU or 11β-MNTDC in Cx rats resulted in favorable changes in body composition and maintenance of BMD comparable to those of T.
ABSTRACT: The objective of this study was to determine whether the rabbit was a suitable model to test new synthetic androgens for potential liver toxicity within a short dosing interval. Adult male rabbits were dosed orally daily on days 0–13 with 17α‐methyltestosterone (MT) as a positive control and testosterone (T) as a negative control to validate this model. Synthetic androgens tested were: 7α‐methyl‐19‐nortestosterone (MENT), dimethandrolone‐undecanoate (DMAU), and 11β‐methyl‐19‐nortestosterone‐17β‐dodecylcarbonate (11β‐MNTDC). Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and sorbitol dehydrogenase (SDH), as well as clearance of intravenous injected bromsulfonphthalein (BSP) from serum on days 0, 7, and 14, were determined. As expected, T (10 mg/kg/d) did not adversely affect BSP retention or serum liver enzymes. MT (10 mg/kg/d) increased BSP retention, and AST, ALT, GGT, and SDH levels, indicating that this model could detect androgens known to be hepatotoxic. DMAU and MENT (10 mg/kg/d) increased BSP retention and all 4 serum liver enzymes as well, but the effects were less than those observed with MT at the same dose. All parameters returned to baseline 2 weeks after cessation of dosing. 11β‐MNTDC at 10 mg/kg/d did not have an effect on BSP retention or liver enzymes, but a slight increase in serum GGT levels was observed in rabbits treated with 25 mg/kg/d. For the androgens that exhibited liver toxicity at 10 mg/kg/d (MT, DMAU, and MENT), a no‐observed‐effect level of 1 mg/kg/d was established. Overall ranking of the synthetic androgens from most to least hepatotoxic on the basis of percent BSP retention was: MT & DMAU > MENT > 11β‐MNTDC. Hence, the rabbit appears to be a promising model for detection of potential liver toxicity by synthetic androgens using BSP clearance and serum liver enzyme levels as early indicators of injury.
Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.
