Presence of immunoreactive gonadotropin releasing hormone (GnRH) and its receptor (GnRHR) in rat ovary during pregnancy

The present study aims at quantification of gonadotropin releasing hormone (GnRH) by radioimmunoassay, relative expression of its mRNA by real-time PCR accompanied by its cellular localization in the rat ovary by immunonohistochemistry (IHC) during different time points of pregnancy. To determine the involvement of endogenous ovarian GnRH in receptor mediated local autocrine/paracrine functions within the ovary, the cell specific localization of the classical receptor for GnRH (GnRHR) in the ovary by IHC and expression pattern of its mRNA were studied during pregnancy. Receptor expression during each time point within the ovary was reconfirmed by Western blot analysis accompanied by densitometric analysis of the signal intensity. Results reveal that the content of ovarian GnRH reaches its maximum on Day 20. The densitometric analysis of GnRHR receptor expression from Western blot study exhibits a decreasing trend by Day 20. Presence of GnRH and GnRHR mRNA in the ovary indicates the local synthesis of both ligand and receptor in the rat ovary. Differential expression of GnRH/GnRHR in the corpus luteum throughout pregnancy strengthens the hypothesis of the involvement of ovarian GnRH in local ovarian functions by receptor-mediated mechanisms. The expression of GnRH and GnRHR in the atretic antral follicles is indicative of the possible involvement of this decapeptide in processes like follicular atresia. The expression of GnRH/GnRHR in the nonatretic antral follicles and their oocytes requires further in-depth investigation. Collectively, this study for the first time reveals the presence of endogenous ovarian GnRH/GnRHR supporting their possible involvement in local autocrine/paracrine functions during pregnancy.     

Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.     

Plasma luteinizing hormone concentrations in cows given repeated treatments or three different doses of gonadotropin releasing hormone

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9 g) insert (CIDR) for 10 d and 500 μg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12 h apart) and 100 μg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40 h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater (P < 0.05) in GnRH38 (8.8 ± 1.1) than in Control (5.1 ± 1.3), with that in GnRH40 (5.8 ± 1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0 ± 0.4) than in either GnRH38 (P < 0.05; 7.0 ± 0.3) or Control (P < 0.09; 7.1 ± 0.4), mean postovulatory P4 (ng/mL) was greater (P < 0.01) in Control (4.2 ± 0.7) than in GnRH38 (2.9 ± 0.6) or GnRH40 (3.0 ± 0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2 mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 μg of GnRH. Cows given 250 μg GnRH released more LH (9.4 ± 1.4 ng/mL) than those given 50 or 100 μg (6.1 ± 1.3 and 5.4 ± 1.4 ng/mL, respectively), and had an LH surge of longer duration than those given 50 μg (6.8 ± 0.4 vs. 5.1 ± 0.3 h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 μg GnRH had a greater pituitary release of LH.     

Effect of intrauterine administration of gonadotropin releasing hormone on serum LH concentrations in lactating dairy cows

The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF_2α given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF_2α-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.     

Effect of clomiphene citrate on pituitary responsiveness to gonadotropin releasing hormone in rams and wethers

The objective of this study was to determine the effect of clomiphene citrate (clomid) on pituitary responsiveness to gonadotropin releasing hormone (GnRH) in rams and wethers. Doses of 200 mg clomid per ram and 1 mug GnRH per 50 kg body weight were used in studies on 12 rams and 4 wethers. The experimental design involved bleeding each animal at 15-minute intervals for 6.5 hours. At the end of the first hour, GnRH was injected IV. The second GnRH challenge was administered 0.5 hours after an injection of clomid or vehicle (4.5% sorbitol solution) which was given on the third hour. The relative response to clomid or vehicle was calculated as the mean increase in concentration of LH during the two-hour period after the second GnRH injection. Each treatment (clomid and vehicle) was given to all animals with a 14-day recovery period between treatment days. The relative response for the rams receiving vehicle (1.80 +/- 0.65) was greater (P < 0.05) than the response during clomid treatment (0.34 +/- 0.22). This suppression of LH response by clomid was observed in 10 of the 12 rams. In contrast to the rams, the concentrations of LH in wethers after the second GnRH injection were lower than those observed after the first GnRH injection. Similar to the rams, the relative response following clomid treatment of wethers (0.04 +/- 0.04) was less than the relative response (P > 0.05) following vehicle (0.40 +/- 0.16). The results suggest that clomid at this dosage inhibits GnRH-induced release of LH from the pituitary of rams but not of wethers.     

Evidence for calcium mediation of gonadotropin releasing hormone action in the pituitary

Despite the relatively long time since the isolation, characterization, and complete chemical synthesis of the gonadotropin releasing hormone (GnRH), very little information has become available which has elucidated the manner by which this hormone evokes gonadotropin release from the pituitary. Recently, a line of evidence has developed which suggests that calcium (Ca²⁺) may play a central role in GnRH stimulation of gonadotropin release from cultured rat pituitary cells.     

Effects of 72 hr calf removal and/or gonadotropin releasing hormone on luteinizing hormone release and ovarian activity in postpartum beef cows

A study was conducted to determine the pituitary and ovarian responses to 72 hr calf removal (CR) and/or gonadotropin releasing hormone (GnRH) in beef cows. Forty-eight Angus, Simmental, and Charolais crossbred cows in moderate body condition were allotted to an experiment of 2 x 2 factorial design involving CR and GnRH. At 30 to 32 days postpartum, calves were removed for 72 hr from the CR and CR plus GnRH groups. All cows were injected (i.m.) with saline or 200 mug of GnRH at 33 to 35 days postpartum. Saline or GnRH was injected 5 hr before calf return. Plasma luteinizing hormone (LH) was measured in blood samples collected every 30 min for 5.5 hr beginning 30 min prior to injection of saline or GnRH. Plasma progesterone was measured in blood samples collected 0, 7, and 14 days after GnRH injection and 7 and 14 days following the first detected estrus. There were no differences (P>0.05) in the interval to peak LH release or the magnitude of the LH release between the GnRH and CR plus GnRH groups; however, the GnRH induced release of LH was greater (P<0.05) over time when preceded by CR. Plasma progesterone concentrations were increased on day 7, compared to day 0, after GnRH injection in 57% and 50% of the animals in the GnRH and CR plus GnRH groups, respectively. However, behavioral estrus was not observed in any of the cows between days 0 and 7 after GnRH injection. A higher (P<0.05) percentage of the cows injected with GnRH formed luteal tissue compared to cows injected with saline; however, the luteal lifespan following GnRH injection was decreased relative to the luteal lifespan following the first observed estrus. The mean interval from calving to first estrus was decreased (P<0.05) by 17 days in the CR group relative to the other groups, and calf removal had no detrimental effect on milk production at 80 days postpartum or on calf weaning weights at approximately 7 months of age. In summary, 72 hr CR decreased the postpartum interval and increased the pituitary responsiveness to GnRH. Pretreatment with 72 hr CR did not alter circulating progesterone concentrations or luteal lifespan of corpora lutea induced by GnRH.     

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.     

Bioactive forms of gonadotropin releasing hormone in the brain of an Indian major carp,Catla catla (Ham.)

Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami ofCatla catla. Gonadotropin releasing hormone activity was studiedin vitro using enzymatically dispersed carp pituitary cell incubation system. Gonadotropin released into the medium was measured by carp gonadotropin-radio immuno assay. Acetic acid extracted hypothalamic material was subjected to acetone fractionation. Among the three protein pellets obtained at different time periods (ACI, ACII and ACIII), AC II exhibited the gonadotropin releasing hormone activity. Gel filtration of AC II through Sephadex G-25 column showed three protein peaks (SG I, SG II SGIII) and only S G II demonstrated strong gonadotropin releasing hormone activity. Elution of SG II through FPLC Mono Q column (an anion exchanger) in NaCl gradient programme showed one unadsorbed (MQ I) and three adsorbed (MQ II, MQ III and MQ IV) protein peaks. MQ III, which was eluted with 51% NaCl, exhibited gonadotropin releasing hormone activity. Surprisingly, unadsorbed fractions, MQ I, also showed gonadotropin releasing hormone activity. MQ 1 was therefore subjected to FPLC Mono S (a cation exchanger) column chromatography where a highly active gonadotropin releasing hormone enriched peak, i.e., MS III, could be eluted with 45% NaCl. These findings show thatCatla catla hypothalamus has two forms of gonadotropin releasing hormones one anionic (carp gonadotropin releasing hormone I) and another cationic (carp gonadotropin releasing hormone II). These two forms of gonadotropin releasing hormones were also active in heterologous carp species, rohu(Labeo rohita), mrigal(Cirrhinus mrigala) and an exotic common carp(Cyprinus carpio). Combined activity of two forms of gonadotropin releasing hormones was significantly greater as compared to any of the single form.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Enhanced biological activity of dimeric gonadotropin releasing hormone

The biological activities of a series of dimeric analogs of des-Gly10-[D-Lys6]GnRH-NHEt cross-linked at Lys6 by malonic acid and elongated by Gly, i.e., HO-Glyn-CO-CH2-CO-Glyn-OH (n = 0, 1, 2), were analyzed in vitro and in vivo. All three dimeric analogs displayed increased activity in receptor binding and in LH release assays than the original monomer, and dimer Ib (n = 1) showed the highest potency in vitro. This compound also showed the highest activity in the in vivo postcoital assay, in which GnRH agonist potency is measured by inhibition of pregnancy. These results indicate that GnRH receptor activation is substantially enhanced by dimerization of the agonist ligand.     

Induction of ovulation in gonadotropin treated gilts with synthetic gonadotropin releasing hormone

Four consecutive trials were conducted to investigate the possibility of controlling the time of ovulation in prepuberal gilts pretreated with PMS and HCG. In trial 1 it was shown that the GnRH analog Hoe 766 was superior to other compounds tested. The following trial revealed that 10 mug of that analog is the optimal dose to elicit an ovulatory response. In trial 3 it was found that the majority (73%) of gilts had started ovulating by 39 h after Hoe 766 injection. Individual gilts started ovulating up to 4 h sooner or up to more than 5 h later. Apparently the ovulatory process of an individual gilt extends over a period of 4 - 5 h. Double insemination of 9 gilts at 34 and 41 h after Hoe 766 resulted in fertilization rates and litter sizes that compared favourably with those of corresponding gilts treated with HCG.     

Inhibition of polyadenylation of mRNA by gonadotropin releasing hormone

The mechanism of extra pituitary inhibitory action of gonadotropin releasing hormone (GnRH) was investigated. Simultaneous injection of GnRH caused dose dependent inhibition of dihydrotestosterone (DHT) induced poly(A) polymerase in the ventral prostate of rat. In addition injection of GnRH to DHT treated animals caused reduced incorporation of 3H-uridine into poly(A)+ mRNA. Since poly(A) segment is known to help in translation of mRNA, it is possible that the inhibitory effect of GnRH is due to the inhibition of polyadenylation of mRNA.     

Gonadotropin releasing hormone therapy in functional infertility of dairy cattle

Effects of gonadotropin-releasing hormone (GnRH) on conception rate was tested in 379 repeat-breeders in nine large dairy herds in Louisiana. Cattle with three or more services were treated intramuscularly with GnRH at the time of artificial insemination. The conception rate for the repeat-breeders treated with GnRH was significantly greater than for the controls (56 vs 40%). Furthermore, repeat-breeders that were treated with GnRH for two consecutive times at insemination resulted in a 53% increase in conception rate over the controls.     

A comparative study of the mechanism of action of luteinizing hormone and a gonadotropin releasing hormone analog on the ovary

The mechanism of action of a gonadotropin releasing hormone (GnRH) agonistic analog ([D-Ala6]GnRH) on the rat ovary has been studied in comparison to similar effects of luteinizing hormone (LH). Stimulation of meiosis resumption in vitro in follicle-enclosed oocytes by both LH and [D-Ala6] GnRH, was blocked by elevated levels of cAMP as demonstrated when either dibutyryl cAMP or the phosphodiesterase inhibitor methylisobutylxanthine was present in the culture medium. In vivo, the prostaglandin synthase inhibitor indomethacin, which blocks LH-induced ovulation, also inhibited ovulation induced by the GnRH analog in hypophysectomized rats. On the other hand, the potent GnRH-antagonist [D-pGlu1, pClPhe2, D-Trp3,6] GnRH which blocked the stimulatory effect of the agonist on oocyte maturation and ovulation had no effect on LH action. It is concluded that while a GnRH-like peptide does not seem to mediate LH action on the ovarian follicles, both LH and GnRH agonist share some common mechanistic pathways at a post-receptor locus.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.