Oestrus, time of ovulation, ovulation rate and conception rate in progestagen-treated ewes given Gn-RH, Gn-TH analogues and gonadotrophins.

The influence of Gn-RH, hCG and a PMSG-hCG mixture (PG600) on the time of ovulation, ovulation rate and on the occurrence of oestrus in ewes treated with progestagen-impregnated sponges for 12 days examined. The effects of Gn-RH analogues on plasma LH, oestrus, ovulation and conception rate were also investigated. Six separate experiments were carried out. When 50 micrograms Gn-RH were given 24 h after sponge removal ovulation occurred in 44--46% of ewes within 24 h and in all ewes by 34 h. Gn-RH was a more potent ovulation synchronizer than hCG. Both hCG and PG600 reduced the incidence of overt oestrus. Gn-RH also had this effect in ewes treated during February and May but not in August and September. Gn-RH analogues given 2 days before sponge removal significantly increased ovulation rate. The display of oestrus was not affected in ewes treated 2 days before sponge removal but was suppressed in 43-69% of ewes treated with an analogue at the time of sponge removal. Ovulation occurred in 50-62% of ewes within 30-35 h of injection of Gn-RH analogues, regardless of the time of their administration. The release of LH in response to one analogue was not influenced by the presence of the progestagen-impregnated sponge in the vagina. When given a Gn-RH analogue 2 days before sponge removal or at the time of sponge removal 63 and 62% of mated ewes became pregnant compared with 70% of control ewes.     

Clinical uses of gonadotropin-releasing hormone analogues.

Gonadotropin-releasing hormone (Gn-RH) analogues are synthetic derivatives of the native hypothalamic peptide with alterations in their chemical structure that result in changes in biologic activity. Several Gn-RH agonists are available for clinical use, and all act through the same mechanism: first to stimulate and then to inhibit gonadotropin and gonadal steroid secretion by downregulating the pituitary Gn-RN receptors. This review should provide clinicians with a working knowledge of the physiologic and pharmacokinetic features of Gn-RH agonists. Although over 2000 articles concerning Gn-RH analogues have been published I chose to review only those that were the first to report a novel clinical application. Gn-RH agonists have proved to be extremely efficacious in treating gonadal steroid-dependent problems such as endometriosis, uterine leiomyoma, precocious puberty and prostate and breast cancers, and they have resulted in very few side effects. Long-term use may, however, lead to skeletal calcium loss in women as a consequence of hypoestrogenism. Further research is needed to prevent this and maintain clinical efficacy.     

Conformation of gonadotropin releasing hormone.

The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from F?rster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site.     

Reproductive axis response to repeated lipopolysaccharide administration in peripubertal female rats

Immune system disorders are often accompanied by alterations in the reproductive axis. Several reports have shown that administration of bacterial lipopolysaccharide (LPS) has central inflammatory effects and activates cytokine release in the hypothalamus where the luteinizing hormone releasing hormone (Gn-RH) neurons are located. The present study was designed to investigate the effect of repeated LPS administration on the neuroendocrine mechanisms of control of the reproductive axis in peripubertal female rats (30-day-old rats). With this aim, LPS (50 μg/kg weight) was administered to the animals during 25, 27 and 29 days of age and sacrificed on 30 day of life. Gn-RH, γ?amino butyric acid (GABA) and glutamic acid (GLU), two amino acids involved in the regulation of Gn-RH secretion, hypothalamic content were measured. LH and estradiol serum levels were also determined and the day of vaginal opening examined. The results showed a significant increase in Gn-RH and GLU content (p?<?0.0001), shared by a reduction of GABA one (p?<?0.0001). LH and estradiol serum levels were decreased (p?<?0.01, p?<?0.001) and delay in the day of vaginal opening was also observed in treated animals. Present results show that repeated LPS administration impaired reproductive function, modifying the neuroendocrine mechanisms of control of the axis in peripubertal female rats.     

Induction of fertile oestrus in seasonally anoestrous ewes with low doses of Gn-RH

Oestrus, conception and lambing performance were assessed in progesterone-primed seasonally anoestrous ewes induced to ovulate with gonadotrophin-releasing hormone (Gn-RH), which was administered intravenously for 48 h as either injections of 250 ng at 2-h intervals (n = 15) or as a continuous infusion at the rate of 125 ng/h (n = 12) or 250 ng/h (n = 12).In $\text{14}\text{15}$ of the ewes injected with Gn-RH, a preovulatory LH peak was recorded at a mean time interval of 33.9 ± 1.8 h after the start of treatment. All ewes displayed oestrus and all ovulated, with a mean ovulation rate of 1.67 ± 0.13. Eleven ewes were diagnosed as pregnant and subsequently lambed. Following infusion of Gn-RH, preovulatory LH peaks were recorded in $\text{21}\text{24}$ ewes at a mean time of 36.1 ± 2.9 h (125 ng/h) and 34.7 ± 2.0 h (250 ng/h). All but two of the ewes displayed oestrus and $\text{23}\text{24}$ ovulated. The group mean ovulation rates of 1.27 ± 0.14 (125 ng/h) and 1.75 ± 0.22 (250 ng/h) were not significantly different. Eleven of the 22 ewes mated were diagnosed as pregnant and produced live lambs.These results suggest that fertility of Gn-RH-induced ovulations in seasonally anoestrous ewes is comparable to that apparent in ewes ovulating spontaneously during the breeding season.     

Design and synthesis of C-12 dithiocarbamate andrographolide analogues as an anticancer agent

A series of 21 new analogues of C-12 dithiocarbamate andrographolide was designed and synthesized from natural andrographolide isolated from a common Thai plant, Andrographis paniculata. The reaction used to manipulate the andrographolide scaffold was conducted in one pot under mild reaction conditions. This avoided toxic catalysts and gave nearly quantitative yields of new analogues, generally without by-products and can be easily scaled -up for industrial processing. All new analogues were evaluated against nine cancer cell lines, some analogues exhibited greater selective cytotoxic activity to MCF-7 cancer cell than that of the parent andrographolide and cancer drugs.     

Stimulatory effects of estrogen on gonadotropin-releasing hormone-induced phosphoinositide turnover in granulosa cells.

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide metabolism in granulosa cells by binding to its specific receptor, and suppresses gonadotropin-induced steroidogenesis. Incubation of immature rat granulosa cells with Gn-RH stimulated time-sequential [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI) in a dose-dependent manner; EC50 was at 10 nM. Concurrent exposure to estradiol-17 beta (E2) (100 nM) and Gn-RH (1 microM) augmented 32P-labeling of PI by 5-fold, while Gn-RH alone induced 3.5-fold increase in PI-labeling. In cells preincubated with E2 for 48 h, Gn-RH provoked a 7-fold [32P]phosphate incorporation into PI, suggesting the induction by E2 of Gn-RH-responsible phosphoinositide turnover. E2 alone provoked a low but significant increase in basal labeling rate of PA and PI. Progesterone failed to mimic the action of E2. Essentially similar results were also obtained in mature rat granulosa cells. These results indicate that E2 augments Gn-RH-stimulated phospholipid turnover in granulosa cells, and suggest that estrogens within the microenvironment of the ovary may exert a local autoregulatory effect on their own production pathway through accelerating Gn-RH action to attenuate steroidogenesis.     

Assessment of the sexual and antler potential of the male white-tailed deer (Odocoileus virginianus) by Gn-RH stimulation test

Twelve mature white-tailed bucks were injected with gonadotropin regulating hormone (Gn-RH, 100 micrograms/deer) during the rut (November) and during the spring (April). In the rut, superior bucks (with actual or potential large body weight, trophy antlers and a high social rank) responded to Gn-RH with a small increase of LH (below 20 micrograms/ml) and a profound rise in testosterone (T) (30-50 ng/ml). The inferior animals exhibited high increase of LH (30-40 ng/ml) but a low rise in T (below 10 ng/ml). FSH levels increased only slightly after Gn-RH and the concentrations were not related to reproductive performance. During the spring, increase in LH levels after Gn-RH administration greatly exceeded the rise of T, but no relationship was found between hormonal levels and the reproductive potential. FSH levels increased remarkably after Gn-RH administration. Gn-RH (administered during the rut) might be used for assessment of the potential for reproductive and antler performance.     

Relationship between pituitary responsiveness to Gn-RH and number of Gn-RH-binding sites in pituitary glands of beef cows

Changes in the ability of Gn-RH to induce gonadotrophin release with time after synchronization of oestrus was determined in 4 groups of 6 cows each. Cows were given Gn-RH at 40-min intervals for 6 h beginning at -24, 0, 18 or 36 h (time 0 = removal of progestagen implant). Changes in concentration (ng/ml) of serum LH after Gn-RH averaged 2.9, 6.2, 6.4 and 33.4, whereas serum FSH averaged 25.7, 35.8, 35.8 and 97.3. Thus the responsiveness of the pituitary to Gn-RH had increased by 36 h after implant removal. Other groups of cows subjected to the same synchronization scheme were slaughtered at 0 h, 24 h or at various times after onset of oestrous behaviour. Gn-RH binding to crude pituitary membrane preparations was assessed. There was no apparent change in the affinity constant of Gn-RH-binding sites with time after synchronization. The number of Gn-RH-binding sites remained unchanged until the period of oestrus when a significant decline with time was detected. We conclude that the increase in pituitary responsiveness to Gn-RH that occurs before the preovulatory gonadotrophin surge was not directly associated with changes in number or affinity of pituitary Gn-RH-binding sites in crude pituitary membrane preparations.     

Induction of ovulation in seasonally anoestrous ewes by continuous infusion of low doses of Gn-RH

Two groups of 12 seasonally anoestrous ewes were infused with Gn-RH at the rate of 125 or 250 ng/h for 48 h. Four control ewes were infused with the saline vehicle alone. Mean LH concentrations increased significantly in response to Gn-RH infusion and were significantly higher (P less than 0.05) in ewes receiving 250 ng Gn-RH/h. LH concentrations remained unchanged in the control ewes. Oestrus was detected in 22/24 Gn-RH-treated ewes and occurred at a mean time of 37.0 +/- 1.2 h after the start of infusion. Ovulation occurred in all but one of the 24 Gn-RH-treated ewes with mean ovulation rates of 1.27 +/- 0.14 (125 ng-Gn-RH/h) and 1.75 +/- 0.22 (250 ng Gn-RH/h). These results demonstrate that a sustained elevation in mean circulating concentrations of LH induced by continuous administration of Gn-RH is sufficient to invoke the final phases of follicular development, and thereby ovulation, in the seasonally anoestrous ewe.     

The hormonal and clinical response of gilts with delayed puberty to GnRH: A preliminary study

Four gilts with delayed puberty were treated with 1 mg of Gn-RH intravenously. Three of the four gilts showed heat after treatment and heats recurred thereafter at normal intervals in these three pigs. All pigs responded with increased blood content of LH after injection of Gn-RH (1.45–6.1 ng/ml). The failure of one pig to exhibit heat after the injection of Gn-RH could not be explained by the resulting LH response.The results of this limited trial indicate that one cause of delayed puberty in gilts may be an insufficient gonadotropic release. Further studies are required to elucidate the reasons for the difference in sexual and ovulatory responsiveness of gilts with delayed puberty to Gn-RH.     

Pattern of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in bovine blood plasma after injection of a synthetic gonadotropin-releasing hormone (Gn-RH)

A synthetic gonadotropin-releasing hormone (Gn-RH) was administered to female and male adult bovines in order to study the release of luteinizing and follicle-stimulating hormones into blood by the pituitary gland. Plasma LH and FSH were determined by means of a radioimmunological method. In females as well as in males, increasing doses of Gn-RH (range 50 to 1500 μg) administered i.v. or i.m. caused a linear increase in plasma LH. The release of FSH evidently was curvilinear over the same dosage range.After 2 or 3 injections of Gn-RH every 3 hours, or every 24 hours or more, smaller amounts of LH were released; repeated treatment did not result in reduction of FSH. Thus pituitary depletion of LH occurred more readily than FSH. The effect of Gn-RH on plasma levels of LH and FSH at various stages of the estrous cycle shows a tendency for an increasing release of both gonadotropins on Days 17 – 18 in comparison to Days 4 – 5 or Days 11 – 12.The results suggest that, within the limits allowed by the heterogenous FSH assay and the method of administration used in these experiments, synthetic Gn-RH does not evoke completely normal physiological responses. Therefore, further work is needed to determine its role in improving reproductive function.     

Short-term relationships between plasma LH, FSH and progesterone concentrations in post-partum dairy cows and the effect of Gn-RH injection

Jugular venous blood samples were obtained from 7 dairy cows every 10 min for 10-19 h during the early- or mid-luteal phase of the oestrous cycle, and each cow was given 1 or 2 i.v. injections of 100 micrograms synthetic Gn-RH. Four of these cows were also sampled in a different cycle with no treatment being administered. Peaks of plasma LH, FHS and progesterone were detected in each animal in the absence of treatment; those of LH and progesterone often occurred in parallel. Injection of Gn-RH was always followed by a significant increase in plasma LH and progesterone concentrations and in most cases by a significant FSH increase. There was a significant temporal relationship between the peaks of all 3 hormones. A further 8 cows were sampled during the first 10 days post partum when the mean plasma progesterone concentration was low. An i.v. injection of 200 micrograms synthetic Gn-RH was given to each animal and this resulted in a significant increase in plasma LH and FSH concentrations, but in only one cow was the Gn-RH injection followed by a significant increase in plasma progesterone concentration. The LH response to Gn-RH injection was significantly less in cows injected on or before Day 5 post partum than in cows injected on Days 7-10 post partum.     

Thyroxine treatment prevents the development of photosensitivity in european starlings (Sturnus vulgaris)

Summary In starlings, the breeding season is terminated by a state of photorefractoriness. Birds remain completely reproductively inactive as long as long days are maintained, and only exposure to short days restores photosensitivity. Two experiments investigated the role of different doses of thyroxine in the development of photosensitivity in castrated starlings. First, photorefractory castrated male starlings were moved from long (18L:6D) to short (8L:16D) days, and received in the drinking water either 1 or 10 mg · 1^-1 thyroxine for the first 7 weeks of a 14-week observation period. Control birds regained photosensitivity after 5 weeks of short days, as signaled by a spontaneous increase in plasma LH, whereas the return to photosensitivity was delayed until weeks 7 and 9 in the 1- and 10-mg · 1^-1 thyroxine-treated birds, respectively. In the second experiment, the effect of different doses of thyroxine was explored at the level of the hypothalamic Gn-RH neurosecretory neurones. The acquisition of photosensitivity in control birds transferred from long to short days was characterized by a marked increase in hypothalamic Gn-RH content (while long-day controls maintained low Gn-RH content). Doses of 10 and 20 mg · 1^-1 of thyroxine completely prevented the return to photosensitivity, as seen through changes in either plasma LH concentrations or hypothalamic Gn-RH content, while a dose of 1 mg · 1^-1 allowed a partial recovery of photosensitivity, as hypothalamic Gn-RH content increased to an intermediate level and the spontaneous rise in plasma LH occurred slowly but steadily.Abbreviations Gn-RH gonadotrophin-releasing hormone - LH luteinizing hormone - LHRH-I luteinizing hormone releasing hormone     

Plasma concentrations of FSH and LH in entire and castrated prepubertal bull calves treated with Gn-RH.

In bulls there was no increase in plasma FSH and only a small increase in LH over the first 14 weeks of age. In steers (castrated) plasma LH and FSH were unchanged for the first 3 weeks but increased significantly at 7 and 14 weeks. After 100 micrograms Gn-RH, LH release in bulls was minimal until 7 and 14 weeks and there was no comparable rise for FSH. LH and FSH responded to Gn-RH throughout the trial in the steers. The neonatal calf testes selectively inhibited the release of FSH from the pituitary even when challenged with Gn-RH.     

Effects of a systematic application of human chorionic gonadotropin, gonadotropin-releasing hormone analog and bovine anterior pituitary gonadotropin in cows with cystic ovarian disease

Fourty-one cows with ovarian follicular cysts (cysts) diagnosed by rectal palpation and observation of estrus behavior were injected intramuscularly (i.m.) with 20,000 IU of human chorionic gonadotropin (HCG). Ten days after this treatment all the cows were examined per rectum for changes in the ovaries with special regard to luteinization of cysts. Cows not responding to HCG were administered 500(10)mug of an analog of gonadotropin-releasing hormone (Gn-RH), Des-Gly-LH-RH-ethylamide, i.m.. If the cysts remained unchanged 10 days subsequent to the second treatment, a third treatment, 400 KE of bovine anterior pituitary gonadotropin (APG), was given i.m. HCG was clinically effective in only 8 cows (20 %). All of the 8 cows conceived. Of 33 cows not responding to HCG, 18 cows (55 %) responded to Gn-RH analog and 12 cows (36 %) conceived. Eleven (73 %) of 15 cows failing to respond to Gn-RH analog were successfully treated with APG and 6 cows (40 %) conceived. In 37 cases, treatment effects were also evaluated by determining serum levels of progesterone prior to and 10 days subsequent to each treatment. When effects of treatment were judged by 1.0 ng/ml or more increase of serum progesterone levels 10 days after treatment, HCG was effective in 13 of 37 cows (35 %), retreatment with Gn-RH analog was successful in 8 of 16 cows (50 %) and APG was ineffective in 3 cows not responding to both HCG and Gn-RH analog. It may be concluded that the therapeutic effect of HCG is disappointing and about half of the cases not responding to HCG were successfully treated with Gn-RH analog. If the cows did not respond to both HCG and Gn-RH analog, they may not respond to APG either.     

Induction of ovulation with chronic intermittent (pulsatile) administration of Gn-RH in women with hypothalamic amenorrhoea

The physiological and pathophysiological basis of hypothalamic amenorrhoea are reviewed as well as the clinical results of chronic intermittent (pulsatile) administration of Gn-RH in the treatment of infertility. Hypothalamic amenorrhoea is considered to be the result of a deficient hypothalamic secretion of Gn-RH. By pulsatile administration of Gn-RH, which is a pre-requisite of normal pituitary gonadotrophic function, deficient endogenous Gn-RH is replaced. If an adequate dose of Gn-RH is provided, which takes into account the degree of impairment of hypothalamic function in the individual case, follicular maturation, ovulation and corpus luteum formation are achieved in nearly every treatment cycle. Although dependent also on factors other than the treated dysfunction, a high conception rate is achieved.     

Isosteres of natural phosphates. 6. The preparation and properties of Lysophosphotidic acid

We herein report the first chemical synthesis of phosphonic acid analogues of lysophosphatidic acid. The racemic isosteric analogues, 4-acyloxy-3-hydroxybutyl-1-phosphonic acids, of lyso-phosphatidic acid were prepared by both catalytic and hydride reductions of the 4-acyloxy-3-oxobutyl-1-phosphonic acids, a general method for the preparation of the latter having been reported previously. The lysophosphatidic acids have been found to substrates for lysophosphatidic acid acyl transferase, and may be acylated chemically to yield phosphotidic acids. The latter reaction is of use in the preparation of differentially acylated phosphatidic acids.     

Structure-activity relationship studies on the inhibition of the bacterial translation of novel Odilorhabdins analogues

A structure–activity relationship (SAR) study of NOSO-95179, a nonapeptide from the Odilorhabdin class of antibacterials, was performed by systematic variations of amino acids in positions 2 and 5 of the peptide. A series of non-proteinogenic amino acids was synthesized in high enantiomeric purity from Williams’ chiral diphenyloxazinone by highly diastereoselective alkylation or by aldol-type reaction. NOSO-95179 analogues for SAR studies were prepared using solid-phase peptide synthesis. Inhibition of bacterial translation by each of the synthesized Odilorhabdin analogues was measured using an in vitro test. For the most efficient analogues, antibacterial efficacy was measured against two wild-type Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) and against an efflux defective E. coli strain (ΔtolC) to evaluate the impact of efflux on the antibacterial activity.     

Application of the McMurry coupling reaction in the synthesis of tri- and tetra-arylethylene analogues as potential cancer chemotherapeutic agents

Structural redesign of selected non-steroidal estrogen receptor binding compounds has previously been successful in the discovery of new inhibitors of tubulin assembly. Accordingly, tetra-substituted alkene analogues (21–30) were designed based in part on combinations of the structural and electronic components of tamoxifen and combretastatin A-4 (CA4). The McMurry coupling reaction was used as the key synthetic step in the preparation of these tri- and tetra-arylethylene analogues. The structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The ability of these compounds to inhibit tubulin polymerization and cell growth in selected human cancer cell lines was evaluated. Although the compounds were found to be less potent than CA4, these analogues significantly advance the known structure–activity relationship associated with the colchicine binding site on β-tubulin.