Potent GnRH Agonist Research Articles

Kisspeptin Modulates Luteinizing Hormone Release and Ovarian Follicular Dynamics in Pre-pubertal and Adult Murrah Buffaloes.

Kisspeptin is a neuropeptide that governs the reproductive axis upstream to GnRH. We wanted to study whether kisspeptin modulates plasma LH and FSH levels and ovarian follicular dynamics in buffaloes and whether kisspeptin can be used for fixed time artificial insemination (FTAI). We carried out these studies in comparison with buserelin, a potent GnRH agonist. Kisspeptin dose-dependently increased plasma LH levels. However, the kisspeptin-induced increase in LH was short-lived as the peak reached in 15–30 min returned to basal values by 1–2 h. The kisspeptin-induced increase in LH level was less compared to buserelin-induced increase in LH level which sustained over time. Kisspeptin did not enhance FSH release while buserelin resulted in a gradual increase over time. LH response to repeated injections of kisspeptin was greater than that induced by buserelin. While buserelin induced an increase in the number of follicles, kisspeptin induced an increase in the growth rate of the follicle. In adult cycling animals, while both the drugs increased plasma LH levels, the increase was greater in buserelin group compared to kisspeptin group. In contrast to the findings in pre-pubertal animals, kisspeptin induced an increase in both the number as well as the size of follicles compared to buserelin. Our studies on oestrus synchronization, using either kisspeptin-PGF2α-kisspeptin protocol or buserelin-PGF2α-buserelin Ovsynch protocol on day 0, 7, and 9, respectively, revealed that kisspeptin increased the number of follicles at wave emergence and the diameter of dominant follicle after 2nd dose of drug, the oestrus response rate and duration of oestrus, compared to buserelin. However, conception rate was not significantly different among the groups. From our studies, it appears that Kp and Buserelin differentially modulate follicular dynamics depending on the reproductive age of the animals.However, studies in a larger herd are required to confirm whether kisspeptin can be used for oestrous synchronization in buffaloes.

Immunolocalization of Androgen Receptor in the Boar Epididymis: the Effect of GnRH Agonist Deslorelin

Epididymides from nine crossbred male pigs [Polish Landrace x (Duroc x Pietrain)] (n = 3 per each group) were used in this study to show whether there are any differences between androgen receptor (AR) distribution along epididymal duct of a GnRH agonist deslorelin-treated boars when compared to the control tissues. The active agent was administered by way of a subcutaneous controlled-release implant containing 4.7 mg deslorelin at 91 or 147 days of age respectively. Boars from two experimental groups and the control group were slaughtered at 175 day of age. Immunolocalization was performed using a polyclonal rabbit antiserum against the AR. In control boars, strong staining for AR was detected in nuclei of the epithelial (principal and basal) and stromal cells, whereas in boars treated with deslorelin the staining was confined to the principal cell nuclei. In those treated for 84 days, AR-immunostaining was weak or the principal cells were negative for the AR. Irrespective of the time from deslorelin insertion all stromal cells were immunonegative. The results demonstrate for the first time the effect of deslorelin on the distribution of the AR in the three regions of the boar epididymis. It is likely that stromal cells are more sensitive than epithelial cells to the regulation of AR expression by androgen. The morphological and functional alterations along the epididymal duct and lack of spermatozoa within the lumen after deslorelin treatment indicate that a potent GnRH agonist is likely responsible for an impairment of the microenvironment created by epididymal cells for sperm maturation and their storage.

Effects of pregnancy on pulsatile secretion of LH and gonadotrophin-releasing hormone-induced LH release in sheep: a longitudinal study

Pulsatile LH secretion and its control throughout pregnancy have not been fully determined in sheep. Expt 1 determined the patterns of LH secretion in five ewes on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. Mean (+/- SEM) concentrations of LH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on day 60 (0.19 +/- 0.3 ng ml(-1)) and on day 120 (0.18 +/- 0.4 ng ml(-1)) of pregnancy than on day 10 of the oestrous cycle (0.55 +/- 0.04 ng ml(-1)). This decrease was due to a significant reduction in the number and the amplitude of LH pulses. Only on day 120 of pregnancy were progesterone concentrations higher (P < 0.01) than on day 10 of the oestrous cycle. Although concentrations of progesterone on day 10 postpartum were barely detectable, mean LH concentration (0.45 +/- 0.09 ng ml(-1)) was not different from that on day 10 of the oestrous cycle. Expt 2 examined the LH responses in a separate group of four ewes to a physiological dose of GnRH (0.2 microg) on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. The area under the LH response curve and the maximum LH concentrations induced by GnRH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on days 60 and 120 of pregnancy than on day 10 of the oestrous cycle, but these parameters were not different between day 10 postpartum and day 10 of the oestrous cycle. Expt 3 examined the LH responses in a separate group of four ewes to a potent GnRH agonist, buserelin (0.5 microg), on days 10, 60 and 120 of pregnancy. The area under the LH response curve and the maximum LH concentrations induced by GnRH were lower (P < 0.01) on days 60 and 120 than on day 10 of pregnancy, but were not different between days 60 and 120. This longitudinal study demonstrates that the pulsatile LH release and pituitary responsiveness to GnRH decreases progressively as pregnancy advances, but does not support the hypothesis that high concentrations of progesterone are solely responsible for the inhibition of pulsatile LH secretion and GnRH-induced LH release during pregnancy in sheep.

Effects of pregnancy on pulsatile secretion of LH and gonadotrophin-releasing hormone-induced LH release in sheep: a longitudinal study

Pulsatile LH secretion and its control throughout pregnancy have not been fully determined in sheep. Expt 1 determined the patterns of LH secretion in five ewes on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. Mean (+/- SEM) concentrations of LH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on day 60 (0.19 +/- 0.3 ng ml(-1)) and on day 120 (0.18 +/- 0.4 ng ml(-1)) of pregnancy than on day 10 of the oestrous cycle (0.55 +/- 0.04 ng ml(-1)). This decrease was due to a significant reduction in the number and the amplitude of LH pulses. Only on day 120 of pregnancy were progesterone concentrations higher (P < 0.01) than on day 10 of the oestrous cycle. Although concentrations of progesterone on day 10 postpartum were barely detectable, mean LH concentration (0.45 +/- 0.09 ng ml(-1)) was not different from that on day 10 of the oestrous cycle. Expt 2 examined the LH responses in a separate group of four ewes to a physiological dose of GnRH (0.2 microg) on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. The area under the LH response curve and the maximum LH concentrations induced by GnRH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on days 60 and 120 of pregnancy than on day 10 of the oestrous cycle, but these parameters were not different between day 10 postpartum and day 10 of the oestrous cycle. Expt 3 examined the LH responses in a separate group of four ewes to a potent GnRH agonist, buserelin (0.5 microg), on days 10, 60 and 120 of pregnancy. The area under the LH response curve and the maximum LH concentrations induced by GnRH were lower (P < 0.01) on days 60 and 120 than on day 10 of pregnancy, but were not different between days 60 and 120. This longitudinal study demonstrates that the pulsatile LH release and pituitary responsiveness to GnRH decreases progressively as pregnancy advances, but does not support the hypothesis that high concentrations of progesterone are solely responsible for the inhibition of pulsatile LH secretion and GnRH-induced LH release during pregnancy in sheep.

Pluronic ® F127 gel formulations of Deslorelin and GnRH reduce drug degradation and sustain drug release and effect in cattle

The objective of this study was to compare the effectiveness of intramuscular sustained release Pluronic ® F127 (PF127) gel formulations of deslorelin, a potent GnRH agonist, and GnRH to their solution formulations in inducing the release of luteinizing hormone and formation of luteal tissue in cattle. Injectable gel formulations of deslorelin and GnRH were prepared using Pluronic ® F127 (25%, w/w), a block copolymer. PF127 gels sustained the in vitro release of deslorelin as well as GnRH at similar rates and reduced drug degradation in muscle tissue when compared to the solution formulations. Deslorelin, as well as GnRH, elicited desirable elevations in plasma LH and progesterone concentrations in vivo. When compared to the solution formulations, the gel formulations of both drugs induced a broader peak of LH. Also, the peak LH levels were lower and the peak times were delayed with the gel formulations compared to the solution formulations. While the solution dosage form of deslorelin and GnRH elicited similar responses, the PF127 gel formulation of deslorelin induced peak LH levels at an earlier time (3 h for deslorelin versus 5.25 h for GnRH). The results indicate that, deslorelin exerts a pharmacological effect in cattle. The LH response to deslorelin as well as GnRH can be altered by controlling the input or the release rate of the drug. PF127 gel formulations can sustain peptide release and reduce peptide degradation.

Restoration of LH output and 17β-oestradiol responsiveness in acutely ovariectomised holstein dairy cows pre-treated with a GnRH agonist (deslorelin) for 10 days

The objectives of the study were firstly to identify the role of the ovary in maintaining plasma luteinising hormone (LH) concentrations in cows treated with an implant of a potent GnRH agonist (deslorelin), and secondly to characterise the changes in LH following ovariectomy (OVX) in the same animals. Oestrus was synchronised in mature Holstein dairy cows and deslorelin implants were inserted 17 days later into two-third of the cows. A further 10 days later (day 0) all cows had bilateral OVX performed. A control group (CON; n=4) received no treatment and had blood samples collected at 15-min intervals for 8 h on the day prior to OVX (day −1) and similarly on days 4 and 10. One group (DES_IN; n=4) had implants in place for the duration of the study while another group had implants removed (DES_OUT; n=4) at the time of OVX. DES_IN cows were sampled hourly at each sampling session (days −1, +4 and +10), whereas DES_OUT cows were sampled similarly to CON except on day −1 when hourly samples were collected. Predictable post-operative increases in mean LH (0.61 ng/ml versus 1.79 ng/ml; P<0.01) and LH pulse amplitude (0.66 ng/ml versus 1.56 ng/ml; day −1 versus day +10; P<0.01) occurred after CON cows were ovariectomised. Smoothed LH means showed a delayed effect of time compared to arithmetic means. Pulse frequency was unchanged following OVX in CON cows. A comparison of all cows that had been treated with deslorelin from day −1 showed a significant elevation of smoothed mean LH compared to untreated cows (0.80 ng/ml versus 0.34 ng/ml; DES_IN and DES_OUT versus CON; P<0.05). DES_IN cows had a 54% reduction in mean LH from day −1 to +4 following OVX (1.05 ng/ml versus 0.48 ng/ml; P<0.01) indicating the probable involvement of the ovary in the maintenance of elevated basal LH. No further reduction was detected by day +10. The LH response to an intramuscular (IM) injection of 500 μg 17β-oestradiol (E2) on day +11 varied significantly between treatment groups ( P<0.01). CON cows showed a typical LH surge, reaching maximum concentrations (10.3 ng/ml) at 17.3 h post-injection. Even though low amplitude LH pulsatility had been restored in DES_OUT cows by day +4, there was an inconsistent response to E2 on day +12; one cow had an apparently normal surge yet, others showed only attenuated responses. Pulse amplitude in DES_OUT cows was lower at days +4 and +10 compared to CON ( P<0.05). DES_IN cows did not produce any surge after E2. Mean LH prior to OVX (day −1) remained unchanged following the 500 μg oestradiol injection (0.38 ng/ml versus 0.45 ng/ml pre-E2 versus post-E2 compared to 1.05 ng/ml pre-OVX). The results of this experiment implicated ovarian involvement in maintaining elevated basal LH output in cows that were chronically treated with a GnRH agonist. Individual cows varied in their LH surge response to exogenous E2 given 12 days after implant removal, even though LH pulse amplitude and frequency had been restored.

Reproductive responses of early postpartum dairy cattle to continuous treatment with a GnRH agonist (deslorelin) for 28 days to delay the resumption of ovulation

An experiment was conducted to investigate the potential of chronic delivery of a potent GnRH agonist (deslorelin) via subcutaneous implants to delay the resumption of ovulatory cycles in postpartum dairy cattle. Cows received either a single deslorelin implant ( n=40; DES) within 7 days of calving or were untreated ( n=24; CON). Blood samples were collected thrice weekly during the period the implants were in place. Plasma concentrations of progesterone (P4) and 17β-oestradiol (E2) were measured along with selected serum metabolites. Implants were removed after 28 days and cattle monitored daily for behavioral oestrus. Serial weekly blood samples were collected to detect the occurrence of ovulation. Cows were artificially inseminated as they were detected in oestrus from 30 days after implant removal. Pregnancy status was subsequently determined by manual palpation of uterine contents at strategic intervals. Insertion of implants induced ovulation in 3/40 cows as determined by a rise in progesterone 7 days later. Deslorelin implants delayed the onset of ovulatory cycles compared with untreated herdmates (mean 43.4±4.2 versus 57.3±1.6 days postpartum; P<0.001). A noticeable delay of at least 12 days was observed between implant removal and the first animals ovulating. Mean plasma E2 concentrations during the period the implants were in place were similar for DES and CON cows that experienced a prolonged spontaneous postpartum anoestrus (low P4 >60 days), although both groups had concentrations only 20% of CON cows that had ovulated prior to 30 days postpartum. The patterns of recovery following implant removal were highly variable. A number of DES cows showed a low and transient rise in plasma progesterone around 21 days after implant removal. Some cows displayed oestrus but did not appear to form a fully functional corpus luteum with this phenomenon being more prevalent among DES cows (7 of 37 versus 1 of 21; P<0.05). Overall, significantly more DES cows were detected in oestrus without ovulating compared to CON cows. Final pregnancy rates did not differ between DES and CON groups. The mean time to conception for DES cows was longer (21.2±5.6 versus 41.1±7.4 days, CON versus DES; P<0.01). This difference was not present if the time from first ovulation to conception was compared (50.5±5.3 versus 43.5±9.3 days, CON versus DES; P>0.05). Deslorelin implants provided a reliable method of inducing anoestrus when treatment was initiated prior to 3 days postpartum. A variable pattern of recovery was observed which delayed conception but did not ultimately reduce the final proportion pregnant at the completion of mating. The study demonstrates the potential of GnRH agonists to control postpartum reproductive function to manipulate the fertility of dairy cows.

The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows

The objective of the present study was to determine the effect of level of feed intake of pasture on P4 clearance rates in dairy cows. Twelve non-lactating Holstein–Friesian cows aged 4–9 years were randomly allocated to a restricted or ad libitum group. The ad libitum group had unrestricted access to irrigated pasture, whereas the restricted group had access for only 2 h per day. Each animal was drenched orally twice daily with a chromic oxide capsule to allow daily feed intake to be estimated from faecal output (FO). Endogenous progesterone (P4) production was eliminated by subcutanously implanting a capsule containing 6 mg of a potent GnRH-agonist (deslorelin) into the ear of each animal 3 weeks before inserting a CIDR device containing 1.9 g P4 into the vagina. Two luteolytic PGF2α were given 10 days later. Each device was removed after 11 days and residual P4 measured. Daily plasma samples were assayed for P4. Faecal samples were also taken daily and assayed for pregnanes (FP4M) containing a 20-oxo-, a 20α- or a 20β-OH group with EIAs. The average daily dry matter (DM) intake of pasture was higher for cows in the ad libitum group (15.9 versus 6.3 kg DM, P=0.001). Their plasma P4 concentrations were lower (1.08 versus 1.71 ng/ml, P=0.05), even though the average residual P4 content of the used CIDR devices was not affected by feed intake (1.20 versus 1.25 g, P>0.05). The concentrations of FP4M were not affected by level of feed intake (20-oxo-: 3.3 versus 1.7, 20α-: 3.5 versus 3.7, 20β-: 2.1 versus 3.2 μg/g DM). Daily excretion rates of 20-oxo- and 20α- were higher in ad libitum cows (20-oxo-: 17.8 versus 4.3 mg per day, P=0.05; 20α-: 18.2 versus 8.9 mg per day, P=0.001), but daily yield of faecal 20β- was not affected by feed intake (11.9 versus 8.6 mg per day, P=0.5). These results show that there was a negative relationship between feed intake and plasma P4 concentrations in these CIDR-treated GnRH-downregulated Holstein cows. Concentrations of FP4M were not affected by level of feed intake or FO, but daily excretion rate of FP4M was associated with the volume of faeces.

Effect of FSH infusion on follicle development in GnRH agonist-treated gilts

The aim was to investigate the effect of infusion of purified FSH alone on follicle development in hypogonadotrophic GnRH agonist-treated gilts. Large-White hybrid gilts (n = 12) were treated during the mid-luteal phase and again after 28 days (day 0) with a potent slow releasing GnRH agonist. On day 3, seven gilts were infused for 168 h with 1.5 S1 units oFSH h-1 (equivalent to 1.5 units of bioactivity of NIH-FSH-S1 standard) and blood samples were collected. Ovaries were then recovered and all follicles > or = 1 mm in diameter were dissected and incubated for 2 h in 1 ml Eagle's minimum essential medium. The ovaries were recovered from the remaining five GnRH agonist-treated gilts on day 10 and also from five cyclic gilts during the late follicular phase (controls). Plasma FSH concentrations in GnRH agonist-treated gilts were lower (P < 0.01) than in follicular phase controls, increased (P < 0.001) after 1 h of FSH infusion and reached a plateau similar (P > 0.1) to that of controls after 8 h. Basal LH concentrations were similar (P > 0.1) between GnRH agonist-treated and control gilts and remained unchanged (P > 0.1) throughout the infusion period. GnRH agonist treatment reduced (P < 0.01) basal oestradiol concentrations compared with control gilts. Infusion with FSH alone increased (P < 0.001) plasma oestradiol concentrations after 96 h compared with those before infusion; when the animals were killed oestradiol concentrations were higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH than in controls. This was also apparent by vulval swelling and behavioural oestrus. There were more follicles > or 1 mm in diameter in the GnRH agonist-treated groups than in the controls (184, 153 and 86 per animal; P < 0.01). Infusion with FSH increased the maximum follicle diameter (GnRH agonist: < 4 mm; FSH infused: < 12 mm; controls: < 10 mm) and tended to increase (P < 0.07) the mean number of follicles > or = 6 mm diameter per animal (FSH infused: 53; controls: 21). Total oestradiol production in vitro by follicles > or = 1 mm was higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH and in follicular phase controls than in animals treated with GnRH agonist alone. However, oestradiol and testosterone secretion in vitro per follicle > or = 6 mm in diameter was lower (P < 0.05) in FSH-infused animals than in controls. In summary, although infusion of FSH alone stimulated the growth of multiple follicles of preovulatory size in GnRH agonist-treated gilts, steroidogenic output by individual follicles was impaired.

Suppression of ovarian activity in the gilt and reversal by exogenous gonadotrophin administration.

The aim of the current experiment was to study the regulation of follicle development in the pig using a potent GnRH agonist (GnRH-A) to initially suppress follicle development. Large-White hybrid gilts (n = 8) were treated during the luteal phase with GnRH-A. Four of these GnRH-A treated gilts and four control gilts were given a GnRH bolus on days 14 and 28 after GnRH-A administration or during the luteal phase in control gilts. Blood samples were collected for 10 h for FSH and LH, after which 1500 IU PMSG were administered and the ovaries and uteri recovered 72 h later. A further four GnRH-A treated gilts and four control gilts were slaughtered either 28 days after GnRH-A administration or during the luteal phase respectively, and all follicles > or = 1 mm diameter were dissected. The mean basal plasma FSH level was lower (P < 0.01) in GnRH-A treated than control gilts and showed no response to the GnRH challenge although levels increased (P < 0.01) in control gilts. The mean basal plasma LH levels were similar (P > 0.1) in GnRH-A treated and control gilts. Whilst in GnRH-A treated gilts plasma LH levels showed no response to the GnRH challenge, plasma LH levels were increased (P < 0.01) in control gilts. Pulsatile LH secretion was abolished in GnRH-A treated but not in control gilts. Plasma oestradiol levels were lower (P < 0.001) in GnRH-A treated gilts than in control gilts, but nevertheless both GnRH-A treated and control gilts responded to PMSG with increased plasma oestradiol levels. Treatment with GnRH-A reduced both the mean (2.1 vs. 2.7 mm; P < 0.01) and the maximal follicle diameter (4 vs. 6 mm) and reduced (P < 0.01) the total number of follicles > or = 2 mm diameter compared with control gilts. Administration of PMSG increased both mean follicle diameter (5.1 vs. 4.4 mm; P < 0.01) and maximal follicle diameter (7 vs. 9 mm) and caused a reduction (P < 0.001) in the total number of follicles > or = 2 mm diameter in both GnRH-A treated and control gilts. In summary, this study has demonstrated, for the first time in the pig, that the inhibition of follicle development as a result of pituitary down regulation/desensitisation can be reversed by exogenous gonadotrophin treatment. This model will be a powerful tool with which to investigate the precise regulation of follicle development in the pig.

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-Leu 6,Pro 9NEt]-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 μg/kg, sc) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro 9 NEt]-GnRH, 3 μg/kg, im) 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 postpartum 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.

Evidence that gonadotropin-releasing hormone stimulates gene expression and levels of active nitric oxide synthase type I in pituitary gonadotrophs, a process altered by desensitization and, indirectly, by gonadal steroids.

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3-7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.

THE SELECTIVE-INHIBITION OF VASCULAR-PERMEABILITY FACTOR (VPF) EXPRESSION IN OVARIAN-CARCINOMA CELL-LINES BY GONADOTROPIN-RELEASING-HORMONE (GNRH) AGONIST

GnRH agonists have been found to be clinically useful in several hormone-sensitive conditions, including cancer. However, there is controversy regarding a direct action of these agents on the pathologic tissue of a given disease process, in particular, tumors. In this study, we examined the effects of D-Trp(6) -LHRH, a potent GnRH agonist, on the expression of an increasingly important angiogenesis factor, VPF (vascular permeability factor)/VEGF (vascular endothelial growth factor). Ovarian carcinoma cell lines exposed to D-Trp(6) -LHRH in culture demonstrated a reversible inhibition of VPF mRNA expression and a parallel decrease in the endothelium-specific mitogenic activity of the conditioned media from these treated cultures. This study reports a novel activity of a GnRH agonist and provides a starting point to investigate the in vivo anti-angiogenic properties of GnRH peptide analogs.

Postinsemination Administration of Receptal: Follicular Dynamics, Duration of Cycle, Hormonal Responses, and Pregnancy Rates

In Experiment 1, concentrations of LH, FSH, and progesterone, but not estradiol-17β, in blood serum were increased during 6 to 12h after injection of 8μg of receptal (GnRH agonist) administered on d 11 to 14 after estrus (d 0) and at first AI compared with saline treatment in lactating Holstein cows. Beginning 2 to 3 d after injection of receptal, concentrations of progesterone were increased for 3 d in nonpregnant cows and for 12 d in pregnant cows compared with controls of similar pregnancy status. Number of ovarian follicles determined by ultrasonography during 10 d after receptal was reduced, specifically those with antral diameters of ≥10mm. The dominant follicle in both groups began to decrease in diameter on the day following treatment, but the next dominant follicle began to increase in diameter 2.3±.7 d later in receptal-treated cows than in controls, accounting for an increase in cycle duration of 2.5±.8 d. In Experiment 2, a double-blinded study was conducted in eight herds in which cows (n = 1013) were AI at first detected estrus after 50 d postpartum and assigned randomly to receive either saline or 4, 8, or 12μg of receptal on d 11 to 14 after first AI. Pregnancy rates were improved in one herd at all doses of receptal, but dose responses were inconsistent in remaining herds. A greater proportion of cows given receptal than controls returned to estrus after 24 d. Administration of a potent GnRH agonist altered number and distribution of ovarian follicles, increased cycle duration, and increased concentrations of progesterone without a consistent increase in pregnancy rates.

Treatment with α-helical-CRF( 9–41) prevents the anorectic effect of 17-β-estradiol

The role of corticotropin-releasing factor (CRF) in the anorexia induced by 17-β-estradiol (E 2) has been assessed in castrated female rats that were trained to eat their daily food ration in three separate meals. Each rat was implanted with a permanent guide cannula that was aimed at the right lateral ventricle of the brain. Seven days after the brain surgery each rat was also subcutaneously implanted with an osmotic minipump containing Buserelin, a potent GnRH agonist that induces reversible castration in rats. Eight rats were used in the study, and each of them underwent four experimental treatments that consisted of a) a subcutaneous (SC) injection of oil combined with an intracerebroventricular (ICV) infusion of saline, b) a SC injection of E 2 combined with an ICV infusion of saline c) a SC injection of oil combined with an ICV infusion of a-helical CRF (9–41), and d) a SC injection of E 2 combined with an ICV injection of α-helical CRF (9–41). Subcutaneous injections of E 2 or oil were carried out the day before the ICV infusions of α-helical CRF (9–41) or saline. Intracerebroventricular infusions were performed 30 min before the meal for which the interaction effect of E 2 and a-helical CRF (9–41) on food intake was determined. E 2 and a-helical CRF (9–41) interacted on food intake; E 2 brought about a 33% reduction in food intake in rats when infused with saline, whereas it was without effect when infused with a-helical-CRF (9–41)-treated rats. The present results provide evidence that CRF is involved in the anorectic effect of E 2.

Administering a GnRH agonist (receptal) after insemination fails to improve pregnancy rates at first service (1992)

Two experiments were performed to determine the influence of administering a highly potent agonist of gonadotropin-releasing hormone (Receptal) on various reproductive characteristics in dairy cows. In Experiment 1, lactating Holstein cows were treated with either saline (n = 51) or 8 μg of receptal (n = 50) on d 11 to 14 after estrus (d 0) and first service. Peak concentrations of LH, FSH, and progesterone, but not estradiol-17b, in blood serum were increased during 6 to 12 h after injection of Receptal. Pregnancy rates were unaffected by treatment. Concentrations of progesterone in blood serum were increased in nonpregnant and pregnant cows after injection of Receptal. Return to estrus in Receptal-treated cows increased by 2.5 ± .8 days compared to controls. The number of follicles >10 mm in diameter, assessed by transrectal ultrasonography, were reduced and follicular development was altered after Receptal. In Experiment 2, various doses of Receptal were tested in eight dairy herds, including 1,013 inseminations at first service. Cows were given a single injection of either saline or 4, 8, or 12 μg of Receptal on days 11 to 14 after first service. Pregnancy rates were not improved consistently in all herds and failed to increase across all herds. We concluded that administering a potent GnRH agonist altered number and distribution of ovarian follicles, increased cycle length, and increased concentrations of progesterone, without a consistent increase in fertility.; Dairy Day, 1992, Kansas State University, Manhattan, KS, 1992;

Effects of TAP-144-SR, A Sustained-Release Formulation of a Potent GnRH Agonist, on Experimental Endometriosis in the Rat.

A new, simple experimental endometriosis model was established by auto-transplanting endometrial tissue fragments beneath kidney capsules in female rats. The transplanted endometrial tissue grew well, forming a fluid-filled cyst, which reached maximal size 2 to 3 weeks after transplantation. The growth and maintenance of the transplants was dependent on the ovary: ovariectomy induced regression of well grown transplants. The therapeutic effects of TAP-144-SR (biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent GnRH agonist, TAP-144 (D-Leu6-[des-Gly10-NH2]-GnRH ethylamide, leuprolide acetate) were studied with this rat endometriosis model. A single sc injection of TAP-144-SR (corresponding to 1, 10 or 100 micrograms/kg/day of TAP-144), suppressed the growth of the transplanted endometrial tissues and uterine weight in a dose-dependent manner. At 100 micrograms/kg/day, the suppressive effect was more marked in rats given TAP-144-SR than in those given TAP-144 solution. The extent of suppression was comparable to that caused by ovariectomy. Serum and pituitary concentrations of LH and FSH were also reduced more markedly by the administration of TAP-144-SR than by TAP-144 solution. From these results, the present endometriosis model was found to be useful for the evaluation of compounds with potential therapeutic activity. The sustained-release formulation of TAP-144 seems to be beneficial over its solution in terms of both convenience and efficiency for therapy of patients with endometriosis.

The effect of a potent GnRH agonist on gonadal and sexual activity in the horse

A potent agonist of gonadotropin releasing hormone (GnRHa) was administered to stallions (n=3) and mares (n=4) during the physiologic breeding season in order to downregulate gonadal activity. Stallions received an increasing regimen of 12.5 mg (5 d), 25 mg (10 d), and 50 mg (15 d) GnRHa per day, for a total of 30 d. Libido and semen quality were not affected by the treatment. The only treatment effect observed was an initial rise in luteinizing hormone (LH) concentration and a slight decline in testosterone concentration during treatment in two of three stallions. These data suggest that daily GnRHa therapy is not a practical method for the suppression of sexual behavior in the stallion at the dosage and interval utilized. Mares received 25 mg i.m. GnRHa per day, for a total of 35 d (n=2), or 40 d (n=2). While estrus and ovulation occurred in two mares during the first half of treatment, follicular development was suppressed in all mares by the end of treatment. Following treatment, ovulation occurred in all mares between 12 and 16 d after treatment. While gonadal activity and estrous behavior can be suppressed in mares with GnRHa therapy, animals require up to 30 d of treatment before suppression of ovarian activity is achieved.

Model of the Distribution and Metabolism of the Gonadotropin-Releasing Hormone (GnRH) Agonist [d-Trp6,Des-Gly-NH210]GnRH Ethylamide in Man

The kinetics of plasma immunoreactive [D-Trp6,Des-Gly-NH2(10)]GnRH ethylamide, a potent GnRH agonist, was assessed in nine normal adult men over a period of 24 h after a rapid sc injection of 1 or 10 micrograms peptide/kg BW. The specific RIA of the GnRH agonist is characterized by a minimum detectable dose of 5.0 +/- 0.5 pg peptide, while the midrange effective dose is 73 +/- 8 pg. The within- and between-assay coefficients of variation range from 2-7% and from 10-15%, respectively. When the volume of the central compartment is set proportional to the logarithm of the plasma concentration of the GnRH agonist, the time courses of both doses are statistically well represented by a two-compartment model to which a source compartment is added to represent the sc route of administration. The sc diffusion to plasma is 5 times faster than any other fractional transport rate of the model (5%/min). Disposal occurs at a maximum rate of 0.55%/min from the central compartment, which includes plasma, and 0.25%/min from the peripheral compartment. As simulated with the model, 90% of the steady state is reached 13 h and 45 min after the onset of a sc continuous infusion. A molecule spends, on the average, from 6 h to 9 h and 45 min in the whole organism; 30-50% of this period is accounted for by residence in the central compartment. The volume of the central compartment reaches 208 mL/kg BW, and the MCR is estimated at 1.18 mL/min.kg BW for a steady state of 1 ng/mL plasma. The plasma dynamics of [D-Trp6,Des-Gly-NH2(10)]GnRH ethylamide in men are at least 10 times slower than those reported for the natural GnRH, thus indicating the relevance of its clinical use in suppressive therapy of LH secretion.

Endocrinological Studies on TAP-144-SR, a Sustained-Release Formulation of a Potent GnRH Agonist (D-Leu6-(des-Gly10-NH2)-GnRH Ethylamide), in male Rats.

The paradoxical effects of TAP-144-SR, a biodegradable sustained-release formulation of a potent GnRH agonist (TAP-144, leuprolide acetate) were evaluated in male rats by comparing its potency with that of TAP-144 solution. A single sc injection of TAP-144-SR (equivalent to 0.1 mg/kg/day as TAP-144), prepared by encapsulating the agonist in microcapsules of copoly (DL-lactic/glycolic acid), suppressed serum levels of androgens, and the levels remained suppressed for 4 weeks. The potency of the paradoxical effects of TAP-144-SR was evaluated 4 weeks after treatment by comparing it with that of TAP-144 solution administered daily for 4 weeks. Both daily injections of TAP-144 solution and a single injection of TAP-144-SR (equivalent to 0.02, 0.2 or 2 mg/kg/day as TAP-144) decreased the weight of the testes, prostates and seminal vesicles in a dose-dependent manner in a 4-week assay in male rats. TAP-144-SR was more effective than TAP-144 solution in reducing these organ weights. Serum and pituitary concentrations of LH and FSH and serum testosterone levels were also lower in TAP-144-SR-treated than in TAP-144 solution-treated rats. These results indicate that the paradoxical effects were more extensive upon TAP-144-SR treatment, suggesting that maintaining constant serum TAP-144 levels results in more extensive desensitization of the pituitary and testes. These results also suggest advantages of TAP-144-SR over TAP-144 solution in both efficacy and convenience as an anti-prostatic tumor agent.