Comparative effects of cyproterone acetate or a long-acting LHRH agonist in polycystic ovarian disease

A randomized cross-over study was done to compare the therapeutic efficacy of cyproterone acetate (CPA, 50 mg/day orally) and a depot preparation of the LHRH superagonist (D-Trp6 LHRH 3 mg i.m. once a month) in 10 patients with polycystic ovarian disease (PCO). The two treatment periods were separated by 6 months. Both treatments resulted in marked clinical improvement. In response to CPA treatment, basal plasma gonadotropin, estradiol, estrone, testosterone and androstenedione levels significantly decreased. In response to D-Trp6 LHRH, both basal and stimulated gonadotropin levels were completely suppressed after 3 weeks of treatment. After initial elevation on day 2, plasma ovarian steroid levels fell into the castrate range, without any change in dehydroepiandrosterone sulfate levels. Urinary 3 alpha-androstanediol excretion decreased significantly. In patients with PCO, LHRH-A induced more complete gonadotropin inhibition than did CPA. However, following cessation of either therapy, the disease rapidly recurred.     

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate)

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.     

Histological studies on the therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) in an experimental endometriosis model in rats

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.     

Repeatability of events during spontaneous and gonadotrophin-induced oestrus in bitches.

Oestrus induction using equine chorionic gonadotrophin and human chorionic gonadotrophin was successful in five out of six bitches, although the first day of increased plasma progestagen concentration differed considerably between bitches. Induced oestrous periods differed from spontaneous cycles in the timing of vaginal epithelial cell cornification; plasma oestrogen concentrations were generally greater and progestogen concentrations were less in induced cycles. These results suggest that this schedule of oestrus induction would not be suitable for allowing mating on a predetermined day.     

Induction of fertile oestrus in the bitch using Deslorelin, a GnRH agonist

Oestrus induction in various canine breeds was attempted in 32 bitches. A group of 8 bitches were treated 80–160 d following their previous oestrus (G1) whereas a second group of 24 bitches (G2) were implanted 200–590 d following their previous oestrus. The treatment for each bitch consisted in one Deslorelin implant (Suprelorin®4,7mg, Virbac, France), inserted subcutaneously in the post-umbilical region. Ovulation, pregnancy rate and litter size were recorded. All bitches came in heat 4.3 ± 1.4 d after implantation (2–7 d). Ovulation was reported in 62.5% in G1 and 87.5% in G2. One bitch refused mating and since no AI was performed, she was not considered for further analysis. Pregnancy was obtained in 25% in G1 versus 78.3% in G2. Mean litter size was 6.7 ± 3.5 puppies (1–14). Luteal failure was suspected in 3 bitches, two that remained non-pregnant and one which aborted 58 d post-ovulation since the owner refused progesterone supplementation. Deslorelin implants can therefore be considered as a valuable alternative to induce fertile oestrus in bitches in anoestrus. Follow-up of the luteal phase is recommended, since some bitches might encounter luteal failure.     

Long-term suppression of pituitary-gonadal function with three-month depot of leuprorelin acetate in a girl with central precocious puberty

OBJECTIVE: The efficacy of a 3-month depot preparation of the GnRH agonist leuprorelin acetate in central precocious puberty was studied. METHODS: Treatment with a 3-month depot of leuprorelin acetate was performed subcutaneously in a 7.3-year-old girl with central precocious puberty. RESULTS: During treatment the hormonal suppression was constant and complete as demonstrated by suppressed GnRH stimulation tests and prepubertal estradiol plasma levels. The size and volume of the uterus and ovaries returned to the normal range. The rate of bone maturation was significantly reduced with a ratio deltaBA/deltaCA of 0.58 for 3 treatment years. Thus, the effects of treatment were comparable to those reported for treatment with 1-month depot of GnRH agonists. CONCLUSION: Three-month depots have the advantage of a prolonged injection interval which is more convenient for the patients and reduces costs by necessitating fewer visits to the physician and being approximately 10% cheaper than the 1-month depot. We suggest that comparative and randomized studies be performed to make 3-month depots of GnRH agonists available for routine use in children with central precocious puberty.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

Fertile estrus induced in bitches by bromocryptine, a dopamine agonist: a clinical trial

The dopamine agonist bromocryptine, probably through amplifying gonadotroph (mainly FSH) secretion, was found to be suitable for provoking fertile estrus during the anestrous phase in bitches without functional cycles and/or ovarian activity. We studied estrus induction in 48 bitches after treatment with semisynthetic ergot alkaloid bromocryptine. For habituation a fractional dose of 0.3 mg/bitch was administered for three days followed by larger doses within the range of 0.6 to 2.5 mg/bitch by selecting dose rates on the basis of individual responsiveness and body weight. The long-term daily bromocryptine dose did not exceed 0.6 mg/bitch and 2.5 mg/bitch in small and large sized bitches, respectively. Gradual habituation and individual dose rates have almost completely eliminated the unwanted side effect of emesis. The period between treatment and onset of estrus varied but the average was 19 days. After the onset of estrus bromocryptine administration was usually continued for another 3 to 6 days. Occurrences of estrus, ovulation and pregnancy were monitored by cytological evaluation of vaginal epithelium, rapid ELISA for plasma progesterone and ultrasonography, respectively. Samples for progesterone were taken on Days 7, 9, 12 and 15 and sonograms of ovarian follicles and of fetuses were taken on Days 0, 22 and 35. The bitches involved in the study either regular or irregular cycles. Bromocryptine treatment induced estrus in all of the bitches including 40 of 48 (83%) with ovulation within a regular estrus and 6 of 48 (12.5%) that showed estrus but did not ovulate. Mating or artificial insemination of bitches in their fertile periods twice at two day intervals resulted in an 83% pregnancy rate (40 cases) and 39 (97.5%) of them gave birth to puppies. However, the average litter size was small with 4.8 +/- 1.6 pups.     

Morphine-6-glucuronide, a potent mu agonist

The 3- and the 6-glucuronides of morphine have been examined in binding studies and in vivo. The 3-glucuronide had poor affinity in all binding studies whereas the 6-glucuronide potently labeled mu, but not delta or kappa receptors with affinities similar to morphine. Microinjections of the 3-glucuronide directly into the periaqueductal gray were without effect. The 6-glucuronide, on the other hand, was up to 20-fold more potent than morphine following microinjections in the same region. High doses of the 6-glucuronide produced profound seizure activity. All 6-glucuronide actions were sensitive to the opiate antagonist naloxone.     

Control of oestrus in ewe lambs and yearling ewes with medroxyprogesterone acetate and fluorogestone acetate

Two experiments were conducted in a large flock of Berrichon purebred and Romanov × Berrichon F₁ crossbred ewes. In experiment 1, 157 ewe lambs were placed in six groups according to season of mating and maternal breed. Within each group equal numbers of females were treated with either medroxyprogesterone acetate (MAP) or fluorogestone acetate (FGA)-impregnated sponges for 14 days; dosage of PMSG at the time of sponge removal was 500 and 300 I.U. respectively for pure and crossbred. In experiment 2, 111 dry yearling ewes of the Berrichon breed were involved, of which 35, 51 and 25 had either a short lambing to treatment interval (approximately 3 months), a long lambing to treatment interval (approximately 6 months), or were primiparous or nulliparous. They were also treated either with MAP or FGA.The conception rates were extremely high (overall near 86%) in ewe lambs and not significantly affected by seasons or breeds or treatments. Prolificacy and number of lambs per ewe mated were higher in the F₁ crossbred group than in pure Berrichon females (respectively 1.91 vs. 1.34 lambs/mated ewe). No significant difference between the two progestagens could be observed for any of the productivity criteria. Fertility was equally satisfactory in yearlings (overall near 85%) irrespective of the interval between lambing and mating. However, the fertility of the nulliparous ewes (those that failed to conceive as ewe lambs) was significantly lower (72%). The number of lambs per ewe mated was 1.78 in yearlings with a 6-month interval from lambing to mating but was not significantly different from the numbers recorded in the other two groups (1.37 and 1.36). As in experiment 1, there was no difference in the results obtained after either of the two progestagen treatments.In conclusion, progestagen treatment has been shown in both ewe lambs and yearlings to be a very effective means of increasing lamb production in large flocks. This was achieved in ewe lambs regardless of the season and, in dry yearling ewes, regardless of the interval from lambing to mating in the anoestrous season.     

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.     

Long-term suppression of fertility in female giraffe using the GnRH agonist deslorelin as a long-acting implant

Zoological institutions provide an environment conducive to studying proximate mechanisms influencing reproduction that can provide guidance to both field and captive settings seeking to manage their stock. Both national parks and zoos have space limitations that sometimes require the use of reversible contraception in order to reduce reproductive rate or limit specific individuals from reproducing. We designed a study to test the efficacy of a long-lasting contraceptive in female giraffe by monitoring reproductive endocrinology and behavior. We implanted two animals with the GnRH agonist deslorelin and monitored their endocrine status using fecal steroid analysis. We have previously validated an assay for fecal pregnanes and here we report our validation for fecal estrogens. Both sex steroid concentrations were suppressed in two females, although one female exhibited an immediate post-implantation positive feedback response. Sexual activity nearly disappeared in one animal, whereas the other showed regular sexual behavior. The contraceptive effect lasted for at least 472 d, and successfully suppressed estrous cyclicity in one female for >2 y. We conclude that deslorelin implants provide a minimally invasive means for long-term suppression of reproduction in female giraffe.     

Reversible suppression of pituitary-testicular function by a sustained-release formulation of a GnRH agonist (leuprolide acetate) in dogs

Pituitary-testicular function was studied in 15 dogs following treatment with a sustained-release formulation of a GnRH agonist, leuprolide acetate (LA). Adult male dogs were treated with a single subcutaneous injection of microencapsulated LA (0.1 or 1 mg/kg). Treatment with LA at a dose of 1 mg/kg resulted in decreased (P<0.001) ejaculatory volume and disappearance of morphologically normal spermatozoa within 8 wk and the effect persisted for 6 wk, while the 0.1 mg/kg dose was not adequate to effect suppression of spermatogenesis. The larger dose treatment (1 mg/kg) caused a transient rise in plasma levels of LH and testosterone followed by a marked decline to below the normal level by 2 wk, the low levels being maintained for at least 5 wk, indicating a prolonged effect of LA treatment on pituitary-gonadal axis. Twenty weeks after treatment with LA, a complete return to normal spermatogenesis was observed. The full reversibility of spermatogenesis in the dog after LA treatment suggests that this peptide could be used as a reversible method of male contraception.     

Long-term stimulatory effects of a continuous infusion of LHRH agonist on testicular function in male red deer (Cervus elaphus)

Red deer stags were infused continuously with the LHRH agonist buserelin at 180-270 micrograms/day (1.2-1.8 micrograms/kg/day) for 72 days starting in late winter with the aim of suppressing reproductive function and inducing premature casting of the antlers. Contrary to expectation, the treatment resulted in a long-term stimulation of testicular activity lasting at least 2 months; the increases in plasma concentrations of testosterone were associated with an increase in aggressive behaviour and the development of rutting odour in the urine. The stags cast their antlers at the normal time in spring after the end of the treatments. The results indicate that the pituitary gonadotroph cells in the stag can continue to secrete LH in response to chronic exposure to an LHRH agonist and do not become rapidly desensitized. The effect of the agonist is therefore to cause significant stimulation of testicular activity which is a conspicuous response in the non-breeding season when the stags are already in a hypogonadal state.     

Auxological and biochemical evaluation of pubertal suppression with the GnRH agonist leuprolide acetate in early and precocious puberty

We studied the auxological effects of treatment with the GnRH agonist leuprolide acetate (Lucrin((R))) at 3.75 mg/ 28 days in 38 children with early or precocious puberty. We present our newly developed scoring system, the Puberty Suppression Score (PSS), in which clinical and biochemical parameters determine whether suppression was effective. Leuprolide acetate suppressed pubertal development in the majority of cases. During treatment there was a significant correlation between the number of times that PSS was >0 and gain in predicted adult height (PAH) compared to initial prediction at the start of treatment. After 6 months of treatment, ineffective suppression measured by PSS was associated with the magnitude of gain in PAH. We conclude that a leuprolide acetate dosage of 3.75 mg every 28 days effectively suppresses puberty. PSS is helpful in monitoring the suppressive capacity of a GnRH agonist. We recommend to start with leuprolide acetate at 3.75 mg/28 days and to increase the injection frequency or dose in case PSS is >0 after 6 months of treatment.     

Downward regulation of plasma LH by LHRH agonist, leuprolide acetate, resulting in inhibited renal growth and function in the castrated male rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.     

Bromocriptine-induced premature oestrus is associated with changes in the pulsatile secretion pattern of follicle-stimulating hormone in beagle bitches

The secretory profiles of LH and FSH were investigated before and during the administration of bromocriptine in six beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 6 h every 2 weeks until the next ovulation. Bromocriptine treatment was started 100 days after ovulation. Both before and after bromocriptine treatment, LH and FSH pulses occurred together. The mean duration of the FSH pulse (120 min) was significantly longer than that of the LH pulse (80 min). The interoestrous interval in the bitches treated with bromocriptine was significantly shorter than that of the preceding cycle (160 +/- 3 versus 206 +/- 24 days). The mean basal plasma FSH concentration (7.4 +/- 0.6 versus 6.1 +/- 0.7 iu l-1) and the mean area under the curve for FSH (46.6 +/- 4.7 versus 40.4 +/- 4.4 iu l-1 in 6 h) increased significantly after the start of the bromocriptine treatment. In contrast, the differences in mean basal plasma LH concentration (2.1 +/- 0.2 versus 2.0 +/- 0.2 micrograms l-1) and the mean area under the curve for LH (19.0 +/- 3.1 versus 19.5 +/- 2.5 micrograms l-1 in 6 h) between the day before and 14 days after the start of the bromocriptine treatment were not significant. Bromocriptine administration also lowered the mean amplitude of the FSH pulse and shortened the mean duration of the FSH pulse, without influencing the LH pulse. In addition to demonstrating the concurrent pulsatile secretion of LH and FSH, the results of the present study demonstrate that the bromocriptine-induced shortening of the interoestrous interval in the bitch is associated with an increase in plasma FSH concentration without a concomitant increase in plasma LH concentration. This finding indicates that treatment with the dopamine agonist bromocriptine increase plasma FSH to a concentration that results in the enhancement of follicle development.     

Lasonolide A,a potent and reversible inducer of chromosome condensation

Lasonolide A (LSA) is a natural product with high and selective cytotoxicity against mesenchymal cancer cells, including leukemia, melanomas and glioblastomas. Here, we reveal that LSA induces rapid and reversible premature chromosome condensation (PCC) associated with cell detachment, plasma membrane smoothening and actin reorganization. PCC is induced at all phases of the cell cycle in proliferative cells as well as in circulating human lymphocytes in G₀. It is independent of Cdk1 signaling, associated with cyclin B downregulation and induced in cells at LSA concentrations that are three orders of magnitude lower than those required to block phosphatases 1 and 2A in vitro. At the epigenetic level, LSA-induced PCC is coupled with histone H3 and H1 hyperphosphorylation and deacetylation. Treatment with SAHA reduced LSA-induced PCC, implicating histone deacetylation as one of the PCC effector mechanisms. In addition, PCC is coupled with topoisomerase II (Top2) and Aurora A hyperphosphorylation and activation. Inhibition of Top2 or Aurora A partially blocked LSA-induced PCC. Our findings demonstrate the profound epigenetic alterations induced by LSA and the potential of LSA as a new cytogenetic tool. Based on the unique cellular effects of LSA, further studies are warranted to uncover the cellular target of lasonolide A (“TOL”).