Restart of steroidogenesis in dogs during recrudescence of testicular function following downregulation with a GnRH-agonist implant.

To date, no details are available concerning the restart of steroidogenesis following the downregulation of testicular endocrine and germinative function by gonadotrophin-releasing hormone (GnRH)-agonist implants. This restart was assessed by determining the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase,17,20-lyase (P450c17). The re-establishment of steroidogenesis was initiated by the removal of the GnRH-agonist implant (18.5 mg azagly nafarelin, Gonazon) at 5 months after treatment. Testes were removed at 3-week intervals (weeks 0-24) and four groups were formed according to the stage of spermatogenesis as revealed by the most developed germ cells observed (developmental group [DG] spermatocytes to DG elongated spermatids). Five dogs served as untreated controls. Positive immunostaining for StAR, P450scc and P450c17 was restricted to Leydig cells. Western blot indicated the specifity of the respective antibodies with hints of a expression of canine-specific P450scc and P450c17 proteins. A significant effect of group was observed for a percentage of the immunopositive area (PIA) as an indicator of active Leydig cells for StAR (P<0.05), P450scc (P<0.001) and P450c17 (P<0.001), with PIA being lowest for the DG spermatocytes. With regard to the strength of the immunopositive signal, a significant effect of group was found for P450scc (P<0.01) and P450c17 (P<0.05), with the lowest intensity being observed in DG spermatocytes. At the mRNA level, the upregulation from DG spermatocytes to DG round spermatids was clearly evident but was only significant for P450scc (P<0.05). Thus, downregulation affects the whole cascade of steroidogenesis, whereas withdrawal of inhibition results in a rapid restart, in part indicating a rebound phenomenon.

Development of semen quality following reversible downregulation of testicular function in male dogs with a GnRH agonist implant.

Slow-release GnRH agonist implants have shown to be an effective and reversible alternative to surgical castration. Testicular function is downregulated with an arrest of spermatogenesis on the level of spermatogonia/primary spermatocytes but is fully restored after abolition of downregulation. Aim of this study was to assess the quality of ejaculates after active abolishment of downregulation by implant removal and to follow recrudescence of spermatogenesis. Five dogs - which served as their own controls - were treated with a slow-release implant containing the GnRH agonist azagly-nafarelin. Implants were removed during full downregulation (testosterone <0.1 ng/ml), and attempts to collect ejaculates started from week 4 onwards to week 29. First ejaculates could be obtained between weeks 8 and 12 with the first fully elongated spermatozoa observed in week 10. Volume, %motility and total sperm count increased and %pathomorphology decreased during the course of the study with all ejaculates being in the normal range by week 29. Our data indicate that onset of recrudescence of spermatogenesis coincides with the first testosterone increase after active abolishment of downregulation. Semen quality was fully regained with a significant improvement of %pathomorphology (p < 0.05) and a tendency of improved %motility. However, these observations on an improved semen quality need further validation and no final conclusions can be drawn yet.

Evaluation of the clinical efficacy of Gonazon implants in the treatment of reproductive pathologies, behavioral problems, and suppression of reproductive function in the male dog.

Efficacy of a slow-release gonadotropin-releasing hormone (GnRH)-agonist implant (Gonazon) was assessed in 53 male dogs presented with benign prostatic hyperplasia (BPH), hypersexuality, aggressive behavior (either alone or in combination), excessive micturition, or to suppress fertility. Changes in testosterone (T) and estradiol (E2) concentrations and size of testes and prostate were monitored on Weeks 0, +8, and +26 after implantation. Additional measurements during and after this period were performed in 35 dogs. Clinical signs were assessed by the owners. All implants except one were retained throughout the study. Full downregulation of testicular function (T<0.35 nmol/L) was achieved in 46 dogs, five dogs showed partial downregulation (T = 0.36 to 0.47 nmol/L), one dog did not respond, and another one displayed a transient downregulation on Week +18. On Week +8, mean T and E2 levels were reduced by 96% and 62%, respectively, and did not further decrease. Full downregulation (T<0.35 nmol/L) lasted between 6 to >22 mo in most dogs except two. Compared with pretreatment values, mean testicular and prostatic size was reduced (P<0.00001) by 54% and 52%, respectively, on Week +8 and by 68% and 64%, respectively, on Week +26. Relative reduction of prostatic size was more marked in dogs with BPH than in healthy ones on Week +8 (P<0.05) and Week +26 (P<0.02), and clinical signs of BPH disappeared rapidly after implantation. Dogs affected with BPH were significantly older (P<0.001) than nonaffected ones (9.7 vs. 2.5 yr). Hypersexuality was more common in dogs<3 yr of age, and treatment clearly improved clinical signs. Age significantly affected the response to treatment in aggressive dogs; 75% of the cases responded with an improvement. The only minor and possibly treatment-related events observed were a short-lasting exacerbation of clinical signs of BPH (two dogs), increased weight gain (three dogs), and anxiety (three dogs) with one of these dogs developing a blunt coat. These results demonstrate the clinical efficacy and overall safety of the Gonazon implants.

Controlled delivery of a GnRH agonist by a silastic implant (Gonazon) results in long-term contraception in queens.

Many female cats are spayed to prevent problems associated with calling and unwanted pregnancies. This study describes the safety and efficacy of an alternative approach, using an azagly-nafarelin containing implant (Gonazon) inserted subcutaneously in the neck of six treated queens for 3 years. These six queens together with six controls were permanently housed with vasectomized tom cats, and changes in progesterone concentrations were used to document the contraceptive efficacy of Gonazon. All six control queens ovulated regularly throughout the treatment period (3 years), as shown by regular changes in progesterone concentration. Sixteen ovulatory cycles were observed in each control throughout the study. In Gonazon treated queens, during the week following implant placement, two queens displayed a treatment-induced rise in progesterone concentration. Later on, all treated queens continuously displayed low progesterone concentrations until 3 years post-implant insertion, with the exception of a single isolated episode (at approximately 2.5 years of treatment), of follicular luteinization in two queens. In all queens, azagly-nafarelin concentrations peaked in the week following implant insertion remained high for 1 month and later decreased slowly. After 2.5 years of treatment, azagly-nafarelin concentrations were still greater than 150 pg/ml in 3/6 queens. During a 6-month long extension of the study (36-42 months post-treatment), all queens (treated and controls) were run with intact tom cats. None of them conceived. Following autopsy, ovarian weight and diameter of the uterine horns of 3/6 treated queens were shown to be similar to those of the controls. In conclusion, this study demonstrated that Gonazon efficiently prevented ovulation in queens (100%) for 3 years. Return to fertile heat was not observed towards the end of treatment. However, in half of the treated queens, reversibility of the treatment induced effects on the genital tract was demonstrated.

Recrudescence of spermatogenesis in the dog following downregulation using a slow release GnRH agonist implant.

The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH-agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross-sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig-cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [X(g) (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre-treatment values following implant removal.

Reversible downregulation of endocrine and germinative testicular function (hormonal castration) in the dog with the GnRH-agonist azagly-nafarelin as a removable implant "Gonazon"; a preclinical trial.

Downregulation of anterior pituitary GnRH-receptors by application of a slow release GnRH-implant offers an effective and reversible alternative to surgical castration of the male dog. Aim of the present study was to test the efficacy and the underlying mechanisms of a new non-biodegradable controlled-release device implant (Gonazon((R)), Intervet, containing 18.5mg of the GnRH-agonist Azagly-Nafarelin). Eight male beagle dogs were implanted s.c. at the para-umbilical region. In four dogs implant removal was after 180 days (group 1), in the other four dogs after 365 days (group 2). Eleven weeks after implantation availability of LH was reduced (p<0.0001) by 70%. After an initial increase lasting for about 4 days, testosterone (T) and estradiol (E2) concentrations decreased (p<0.0001) to basal levels within 17.5+/-8.4 days. Size of testes was decreased by about 82% after 17 weeks, size of prostate by about 46% after 5 weeks (p<0.0001). Five to 7 weeks after implantation all dogs were aspermic. Testosterone and estradiol concentrations, together with testicular and prostatic size remained suppressed in all dogs in group 1 and one dog of group 2 until implant removal. The other three dogs of group 2 escaped from down-regulation between 223 and 324 days. Effects on the availability of LH, T, E2 and on testicular and prostatic size were fully reversible after implant removal or escape from down-regulation. In six dogs semen quality was back to pre-treatment values after about 29 weeks, however, one dog developed oligozoospermia while another one stayed azoospermic, probably due to an obstruction within the epididymal duct.

Treatment with a subcutaneous GnRH agonist containing controlled release device reversibly prevents puberty in bitches.

In most species, continuous administration of GnRH agonists desensitizes the pituitary to GnRH, and blocks ovarian function. The aim of this study was to assess the effects of a novel controlled release device containing azagly-nafarelin (Gonazon) to prevent puberty in young Beagle bitches (mean age: 4.88 +/- 0.32 months). Gonazon containing 18.5 mg azagly-nafarelin (n = 10) or a placebo implant (n = 10) was administered subcutaneously. Throughout the 1-year treatment, estrus behaviour was monitored weekly. Plasma progesterone concentrations, as well as body weight and height, were measured monthly. Following implant removal, estrus detection and progesterone measurement were continued until occurrence of puberty in all bitches. Control bitches displayed puberty (estrus, followed by ovulation) at approximately 11.9 +/- 2.7 (range, 8-16) months of age. In contrast, none of the Gonazon treated bitches displayed puberty during the period when Gonazon was present. Following removal of Gonazon, resumption of estrus and ovulation naturally occurred (seven bitches) or was induced (three bitches) approximately 8.5 (1.2-14.3) months later. As a consequence, age of puberty of the Gonazon treated bitches was 25.5 +/- 5 (18-31) months. No clinically detectable side effects were noted in Gonazon treated bitches. Height at withers was unaffected by treatment. Changes in body weight with time were also unaffected by treatment. Implants were well tolerated and generally easy to remove. These data demonstrated that Gonazon safely, efficiently and reversibly prevents reproductive function for 1 year in prepubertal bitches.

Chronic treatment with an agonist of gonadotropin-releasing hormone enhances luteal function in cattle.

Our hypothesis was that luteal function, as determined by plasma progesterone concentrations, and corpus luteum (CL) size is enhanced in cattle administered an agonist of GnRH when the CL is developing as compared with administration of an agonist when the CL is fully functional. Cattle were chronically administered a GnRH agonist, azagly-nafarelin, from Day 3 to Day 21 (D3) or Day 12 to Day 21 (D12) or served as untreated control females (Day 0 = behavioral estrus). Blood samples were serially collected on Days 7 and 14 to evaluate LH secretory patterns and twice daily to measure plasma progesterone. Ultrasonographic examinations were conducted daily to record the area of the CL. CL size and plasma progesterone concentrations were both enhanced in the D3 group as compared with the control group. Progesterone was increased in the D12 group on Days 16 and 17 as compared with the control females. Treatment with GnRH agonist increased basal and mean LH concentrations in both D3 and D12 groups as compared with the controls. We rejected our hypothesis because chronic administration of a GnRH agonist increased plasma progesterone when administered both when the CL was developing and when it was fully functional. The enhanced luteal function was likely due to increased basal LH.

Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists.

Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.