Luteinizing Hormone-releasing Hormone Peptide Research Articles

LHRH-Targeted Redox-Responsive Crosslinked Micelles Impart Selective Drug Delivery and Effective Chemotherapy in Triple-Negative Breast Cancer.

Systemic chemotherapy is efficacious against triple-negative breast cancer (TNBC), but it is often associated with serious side effects. Here, a luteinizing hormone-releasing hormone (LHRH) receptor-targeted and tumor microenvironment-responsive nanoparticle system to selectively deliver chemotherapeutic drugs to TNBC cells, is reported. This delivery system (termed "LHRH-DCMs") contains poly(ethylene glycol) and dendritic cholic acid as a micellar carrier, reversible intra-micellar disulfide bond as a redox-responsive crosslink, and synthetic high-affinity (D-Lys)-LHRH peptide as a targeting moiety. LHRH-DCMs exhibit high drug loading efficiency, optimal particle size, good colloidal stability, and glutathione-responsive drug release. As expected, LHRH-DCMs are more efficiently internalized into human TNBC cells through receptor-mediated endocytosis, resulting in stronger cytotoxicity against these cancer cells than the non-targeted counterpart when encapsulated with paclitaxel (PTX). Furthermore, near-infrared fluorescence and magnetic resonance imaging demonstrate that LHRH-DCMs facilitate the tumor distribution and penetration of payloads in three different animal models of breast cancer, including cell line-derived xenograft (CDX), patient-derived xenograft (PDX), and transgenic mammary carcinoma. Finally, in vivo therapeutic studies show that PTX-LHRH-DCMs outperform both the corresponding nontargeted PTX-DCMs and the current clinical formulation (Taxol) in an orthotopic TNBC model. These results provide new insights into approaches for precise drug delivery of TNBC.

Synthesis and Cytotoxic Study of a Platinum(IV) Anticancer Prodrug with Selectivity toward Luteinizing Hormone-Releasing Hormone (LHRH) Receptor-Positive Cancer Cells.

Platinum drugs including cisplatin are widely used in clinics to treat various types of cancer. However, the lack of cancer-cell selectivity is one of the major problems that lead to side effects in normal tissues. Luteinizing hormone-releasing hormone (LHRH) receptors are overexpressed in many types of cancer cells but rarely presented in normal cells, making LHRH receptor a good candidate for cancer targeting. In this study, we report the synthesis and cytotoxic study of a novel platinum(IV) anticancer prodrug functionalized with LHRH peptide. This LHRH-platinum(IV) conjugate is highly soluble in water and quite stable in a PBS buffer. Cytotoxic study reveals that the prodrug selectively targets LHRH receptor-positive cancer cell lines with the cytotoxicities 5-8 times higher than those in LHRH receptor-negative cell lines. In addition, the introduction of LHRH peptide enhances the cellular accumulation in a manner of receptor-mediated endocytosis. Moreover, the LHRH-platinum(IV) prodrug is proved to kill cancer cells by binding to the genomic DNA, inducing apoptosis, and arresting the cell cycle at the G2/M phase. In summary, we report a novel LHRH-platinum(IV) anticancer prodrug having largely improved selectivity toward LHRH receptor-positive cancer cells, relative to cisplatin.

LHRH-Targeted Drug Delivery Systems for Cancer Therapy.

Targeted delivery of therapeutic and diagnostic agents to cancer sites has significant potential to improve the therapeutic outcome of treatment while minimizing severe side effects. It is widely accepted that decoration of the drug delivery systems with targeting ligands that bind specifically to the receptors on the cancer cells is a promising strategy that may substantially enhance accumulation of anticancer agents in the tumors. Due to the transformed cellular nature, cancer cells exhibit a variety of overexpressed cell surface receptors for peptides, hormones, and essential nutrients, providing a significant number of target candidates for selective drug delivery. Among others, luteinizing hormonereleasing hormone (LHRH) receptors are overexpressed in the majority of cancers, while their expression in healthy tissues, apart from pituitary cells, is limited. The recent studies indicate that LHRH peptides can be employed to efficiently guide anticancer and imaging agents directly to cancerous cells, thereby increasing the amount of these substances in tumor tissue and preventing normal cells from unnecessary exposure. This manuscript provides an overview of the targeted drug delivery platforms that take advantage of the LHRH receptors overexpression by cancer cells.

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.

A phase I study of EP-100, a luteinizing hormone releasing hormone (LHRH) ligand conjugated to a synthetic cytolytic peptide in patients with advanced refractory LHRH- receptor (R)-expressing tumors.

3060 Background: EP-100 is a synthetic 28 amino acid LHRH peptide conjugated to an 18 aa cytolytic peptide. Selective targeting in LHRH-R expressing cells occurs via direct binding to the extracellular membrane receptor followed by disruption of the membrane by the cytolytic peptide portion. A first in human, phase I study was performed. Methods: Eligible pts had adequate organ function and LHRH-R tumor expression, as determined by IHC. EP-100 was given IV (30-60 min) weekly x 3 for cohorts 1- 6 (n=20 at 0.6 - 5.2 mg/m2) and then twice weekly x 3 for cohorts 7- 11 (n=18 at 7.8 - 40 mg/m2) with a week break. The pharmacokinetic profile of EP-100 and antibody production against EP-100 was determined by a validated HPLC/MS and ELISA respectively. Potential activity of EP-100 on the pituitary gonadotropes was determined by plasma LH and FSH. Results: We screened 97 pts, archival tumor tissue obtained from 81 pts; 53 (65%) were positive for LHRH-R, of which 37 were enrolled (female 76%) and treated. The LHRH-R expression rate in breast was 81% (N=21) and in ovarian cancer 56% (n=18). Most pts had breast (n= 16, 42%) ovary (n=7,18%), colon (n=4, 11%) or uterine (n=3, 8%) cancer. Pts were treated in a 3 + 3 standard dose escalation scheme. Doses were escalated 100% in the absence of a grade 2 drug related toxicity. MTD was not reached at the highest dose level of 40 mg/m2. Only one DLT occurred, a grade 2 increase in ALT/AST. The only other drug-related toxicity was a self limited infusion-related skin reaction (grade 1-2) in 10 pts. EP-100 was rapidly cleared from circulation, mean half life ranged from 7.1 ± 3.8 min to 15.9 ± 3.6 min. Best response was stable disease for >12 weeks in 5 pts. In 3 pts rapid, sustained decreases in LH/FSH levels were noted. Antibody production against EP-100 was absent in all pts. Conclusions: The study has completed accrual, the phase II dose is 30-40 mg/m2 twice a week x 3 weeks.EP-100is safe, well tolerated and not antigenic/immunogenic. Preclinical studies indicated synergy with paclitaxel and doxorubicin. A phase II study of EP-100 plus paclitaxel in ovarian cancer is planned.

Abstract 1950: LHRH Peptide decorated cross-linked micelles for the targeted drug delivery to breast cancer

Abstract We have recently developed a series of micellar nanoparticles (NPs) based on linear polyethylene glycol (PEG) and dendritic cholic acids (CA) block copolymer (telodendrimer), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers. The tumor targeting specificity and efficiency of drug-loaded NPs might be further improved by the decoration of cancer cell surface targeting ligands. LHRH (Luteinizing hormone releasing hormone), a natural hormone peptide, is able to bind specifically to the LHRH receptor, which has been reported to be over-expressed in more than 50% breast cancer patients. In the present study, we incorporated the passive targeting of our micellar NPs and the active targeting of LHRH peptide into a single platform with an attempt to further improve the therapeutic index of NPs delivered drugs in the treatment of breast cancer. We synthesized the LHRH peptide on the tentagel resin bead via the solid-phase peptide synthesis method, and demonstrated that human breast cancer cell lines MDA-MB-231 and MDA-MB-435 were able to bind strongly with LHRH peptide beads. Besides breast cancer cell lines, immunohistochemistry staining further demonstrated LHRH peptide was able to specifically bind to human breast tumor tissue as well as transgenic murine mammary carcinoma tissue, but not to normal breast tissue. Alkyne-containing LHRH peptides were successfully conjugated to the azide group at the distal PEG terminus of PEG5k-Cys4-L8-CA8 telodendrimer (cross-linked micelle forming unit) via the “click chemistry”. LHRH peptide ligand significantly enhanced the intracellular delivery of cross-linked micelles in both MDA-MB-231 and MDA-MB-435 breast cancer cells. In addition, the enhanced uptake of LHRH-targeted micelles in those cells can be inhibited by the addition of access of LHRH antibody, indicating the LHRH receptor-medicated internalization. The co-localization of internalized FITC-labeled LHRH-micelles with lysosomal compartment in human breast cancer cells were also observed, suggesting that the endocytosis pathway is likely to be involved in the internalization process. Furthermore, when loaded with paclitaxel (PTX), LHRH-micelles had significantly higher in vitro cellular toxicities against breast cancer cells as compared with the non-targeted micelles. The in vivo biodistribution, tumor targeting specificity and therapeutic efficacies of LHRH targeted micelles in MDA-MB-231 xenograft mouse model are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1950. doi:1538-7445.AM2012-1950

Luteinizing hormone-releasing hormone receptor-mediated delivery of mitoxantrone using LHRH analogs modified with PEGylated liposomes

A sterically stabilized, mitoxantrone-loaded liposome, tailored to target luteinizing hormone-releasing hormone (LHRH) receptor overexpressing cells, was developed to promote the efficiency of intracellular delivery of mitoxantrone through receptor-mediated endocytosis. Liposomes were prepared by lipid film hydration and an ultrasound dispersion process. Thiolated gonadorelin with affinity for the LHRH receptor was chemically coupled to N-[(3-maleimide-1-oxopropyl) aminopropyl polyethylene glycol-carbamyl] distearoyl-l-phosphatidyl-ethanolamine via a thioether bond and subsequently inserted into polyethylene glycol-grafted liposomes. The liposome was characterized in terms of its size, ligand density, drug loading, and leakage properties. The targeting nature and antitumor effects of the liposomes were evaluated in vitro using cultured MCF-7 breast cancer cells. A protein assay of ligand coupling to the liposomal surface indicated that more than 60% of the LHRH peptides were inserted into the liposome bilayer. Up to 1.0 mg/mL of stable liposomal mitoxantrone loading was achieved, with approximately 98% of this being entrapped within the liposomes. In vitro cell culture studies revealed that the gonadorelin-modified liposomes bound to their target cells had significantly higher affinity and better antitumor efficiency than generic drug-loaded liposomes. These events were presumed to occur through specific interactions of the LHRH with its cognate receptors on the cell surface. It was concluded that the targeting properties of the delivery system would potentially improve the therapeutic benefits of mitoxantrone, as compared with nontargeted liposomes.

Novel Polymeric Prodrug with Multivalent Components for Cancer Therapy

We designed, synthesized, and evaluated in vitro and in vivo a novel targeted anticancer polymeric prodrug containing multiple copies of tumor targeting moiety [synthetic luteinizing hormone-releasing hormone (LHRH) peptide, analog of LHRH] and anticancer drug (camptothecin). One, two, or three molecules of the targeting peptide and anticancer drug were covalently conjugated with bis(2-carboxyethyl) polyethylene glycol polymer using citric acid as a multivalent spacer. We showed that LHRH peptide was bound to extracellular receptors and localized in plasma membrane of cancer cells. The designed tumor-targeted prodrug increased the solubility of anticancer drug and offered cytoplasmic and/or nuclear delivery of drug to cancer cells expressing LHRH receptors. The multicomponent prodrug containing three copies of the targeting peptide and drug was almost 100 times more cytotoxic and substantially had enhanced antitumor activity compared with the analogous nontargeted prodrug and prodrugs containing one or two copies of active components.

Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. Invariably the side effects impose dose reduction, treatment delay, or discontinuance of therapy. To limit the adverse side effects of cancer chemotherapy on healthy organs, we proposed a drug delivery system (DDS) with specific targeting ligands for cancer cells. The proposed DDS minimizes the uptake of the drug by normal cells and enhances the influx and retention of the drug in cancer cells. This delivery system includes three main components: (i) an apoptosis-inducing agent (anticancer drug), (ii) a targeting moiety-penetration enhancer, and (iii) a carrier. We describe one of the variants of such a system, which utilizes camptothecin as an apoptosis-inducing agent and poly(ethylene glycol) as a carrier. Luteinizing hormone-releasing hormone (LHRH) was used as a targeting moiety (ligand) to LHRH receptors that are overexpressed in the plasma membrane of several types of cancer cells and are not expressed detectably in normal visceral organs. The results showed that the use of LHRH peptide as a targeting moiety in the anticancer DDS substantially enhanced the efficacy of chemotherapy, led to amplified apoptosis induction in the tumor, and minimized the side effects of the anticancer drug on healthy organs. The LHRH receptor targeting DDS did not show in vivo pituitary toxicity and did not significantly influence the time course or the plasma concentration of luteinizing hormone and its physiological effects on the reproductive functions of mice.

Engineering, cloning, and expression of genes encoding the multimeric luteinizing-hormone-releasing hormone linked to T cell determinants in Escherichia coli

Two synthetic genes were designed and engineered to encode for multimeric luteinizing-hormone-releasing hormone (LHRH) peptides linked to T cell determinants. These genes were cloned into the prokaryotic expression vectors under control of strong inducible promoters, to overexpress the multimeric LHRH peptides as recombinant proteins. Multimeric LHRH–T cell peptides were expressed as insoluble inclusion bodies in Escherichia coli cultures. Cell extracts containing the recombinant proteins showed immunoreactivity on Western blots with monoclonal antibody recognizing the native hormonal peptide. These gene constructs have potential applications in therapy of sex-steroid-hormone-dependent cancers.

Synthetic luteinizing hormone releasing hormone (LHRH) vaccine for effective androgen deprivation and its application to prostate cancer immunotherapy

We have designed a peptide-based immunotherapeutic vaccine for treatment of androgen-responsive prostate cancer. The vaccine targets the luteinizing hormone-releasing hormone (LHRH) decapeptide that results in an androgen-deprivation immunotherapy. The design elements of the peptide immunogens are the LHRH peptide or B cell epitope synthetically linked to different promiscuous helper T cell (Th) sequences, the UBITh ® epitopes, derived from four natural pathogens for effective immunogenicity in outbred populations, and in some cases, also linked to an adjuvanting peptide from Yersinia invasin (Inv) protein. The UBITh ® LHRH immunogens are adsorbed on Alhydrogel or formulated as several different oil-based emulsions and tested in rodents, dogs, and a non-human primate, baboons. The immunogens generate an anti-LHRH antibody response specific to the LHRH decapeptide element in contrast to LHRH conjugate-carrier protein vaccines where only a small portion of the antibody response is directed to the target epitope and epitopic suppression is noted. Individual UBITh ® peptide domains, but not the LHRH and Inv peptide domains, are stimulatory in lymphocyte cultures. The UBITh ® LHRH immunogens in a clinically applicable formulation, controlled the growth of Dunning R3327-H androgen-responsive prostate tumor cells in rats. The results demonstrate universal responsiveness and long duration of androgen deprivation from three diverse species, and thus vaccine efficacy.

Molecular targeting of drug delivery systems to ovarian cancer by BH3 and LHRH peptides

Novel targeted proapoptotic anticancer drug delivery systems were developed and evaluated. Poly(ethyleneglycol) (PEG) conjugates were used as carriers. Camptothecin (CPT) was used as an anticancer agent–apoptosis inductor. Two types of molecular targets were investigated: (1) an extracellular membrane receptor specific to ovarian cancer and (2) intracellular controlling mechanisms of apoptosis. Synthetic peptides similar to luteinizing hormone-releasing hormone (LHRH) and BCL-2 homology 3 (BH3) peptide were used as a targeting moiety and a suppressor of cellular antiapoptotic defense, respectively. Three different conjugates (CPT–PEG, CPT–PEG–BH3 and CPT–PEG–LHRH) were synthesized and examined in A2780 human ovarian cancer cells. Cytotoxicity, expression of genes encoding BCL-2, BCL-XL, SMAC, APAF-1 proteins and caspases 3 and 9, the activity of caspases 3 and 9 and apoptosis induction were studied. Taken together the results indicate much higher cytotoxicity and apoptosis-inducing activity of PEG–CPT conjugates when compared to free CPT. Moreover, the effects of targeted CPT–PEG–BH3 and CPT–PEG–LHRH conjugates were more pronounced than the non-targeted PEG–CPT conjugate. The results confirmed the feasibility of this new two-tier molecular targeting strategy for enhancing the efficacy of cancer chemotherapy.

Targeted proapoptotic LHRH-BH3 peptide.

The purpose of this work was to construct and evaluate a novel targeted proapoptotic peptide for cancer treatment. The peptide consisted of luteinizing hormone-releasing hormone (LHRH) as a targeting moiety specific to LHRH receptors and a synthetic BCL-2 homology 3 (BH3) domain peptide as an apoptosis inducer and a suppressor of antiapoptotic cellular defense. Anticancer activity of the peptide was evaluated on different cancer cell lines. The targeting receptor to LHRH peptide is overexpressed in several cancer cell lines but is not expressed in healthy human visceral organs. LHRH and BH3 peptides when applied separately did not demonstrate cellular toxicity. In contrast, the LHRH-BH3 peptide was toxic in several cancer cell lines. Coincubation of LHRH and LHRH-BH3 peptides significantly decreased cytotoxicity of the latter. It was found that the LHRH-BH3 peptide induced apoptosis by simultaneous inhibition of the antiapoptotic function of BCL-2 protein family and activation of caspase-dependent signaling pathway. The proposed anticancer proapoptotic LHRH-BH3 peptide simultaneously affects two molecular targets: 1) extracellular cancer-specific LHRH receptors and 2) the intracellular controlling mechanisms of apoptosis. The results of this work may be used to design novel approaches for the treatment of various cancers.

Synchronization of Ca(2+) oscillations among primate LHRH neurons and nonneuronal cells in vitro.

Periodic release of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus is essential for normal reproductive function. Pulsatile LHRH release appears to result from the synchronous activity of LHRH neurons. However, how the activity of these neurons is synchronized to release LHRH peptide in a pulsatile manner is unclear. Because there is little evidence of physical coupling among LHRH neurons in the hypothalamus, we hypothesized that the activity of LHRH neurons might be coordinated by indirect intercellular communication via intermediary (nonneural) cells rather than direct interneural coupling. In this study, we used an in vitro preparation of LHRH neurons derived from the olfactory placode of monkey embryos to assess whether nonneuronal cells, play a role in coordinating LHRH neuronal activity. We found that cultured LHRH neurons and nonneuronal cells both exhibit spontaneous oscillations in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) at similar frequencies. Moreover, [Ca(2+)](i) oscillations in both types of cell were periodically synchronized. Synchronized [Ca(2+)](i) oscillations spread as intercellular Ca(2+) waves across fields of cells that included LHRH neurons and nonneuronal cells, although waves spread at a higher velocity among LHRH neurons. These results suggest that LHRH neurons and nonneuronal cells are functionally integrated and that nonneuronal cells could be involved in synchronizing the activity of the LHRH neurosecretory network.

Luteinizing hormone-releasing hormone (LHRH) biosynthesis and secretion in embryonic LHRH.

Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7-10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mM KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide.

Luteinizing Hormone-Releasing Hormone (LHRH) Biosynthesis and Secretion in Embryonic LHRH Neurons

Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7–10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mm KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide.

Midline Nasal Tissue Influences Nestin Expression in Nasal-Placode-Derived Luteinizing Hormone-Releasing Hormone Neurons during Development

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Technical note: recombinant LHRH fusion protein suppresses estrus in heifers.

A recombinant luteinizing hormone-releasing hormone (LHRH) fusion protein was evaluated for its effectiveness in suppression of estrus in heifers. Eight heifers were randomly assigned to two equal treatment groups. Treatments consisted of recombinant ovalbumin-LHRH-7 or recombinant ovalbumin (control). This recombinant chimeric fusion protein consisted of ovalbumin with seven LHRH peptides (ovalbumin-LHRH-7). The plasmid for this protein was expressed in E. coli and was collected and purified as an insoluble protein. One milligram of the respective proteins was suspended in 2 mL of Z-Max adjuvant and administered by intramammary injection three times at 7-wk intervals. Luteinizing hormone-releasing hormone antibody binding was elevated in heifers treated with ovalbumin-LHRH-7 compared to ovalbumin-treated heifers (P < .05). Serum progesterone concentrations (< 1 ng/mL) indicate that the estrous cycle of the four heifers treated with ovalbumin-LHRH-7 was suppressed for a time period ranging from 60 to 238 d, which was different from control heifers (P < .01). Serum progesterone for the control heifers continued to exhibit cyclic profiles over the experimental period. This preliminary study in heifers demonstrated that a chimeric LHRH fusion protein induced elevated concentrations of circulating LHRH antibodies that suppressed estrus for an average of 122 +/- 41 d.

Luteinizing Hormone-Releasing Hormone (LHRH) Neurons Maintained in Hypothalamic Slice Explant Cultures Exhibit a Rapid LHRH mRNA Turnover Rate

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

Double immunogold labelling demonstrating expression of recombinant genes for production of an anti-fertility vaccine

The multiple antibody technique for double immunogold labelling for the simultaneous localization of two antigens with negative staining was utilized to demonstrate the expression of recombinant genes in bacteria, with the primary antibodies being raised in different host species. For the production of a vaccine for immunological control of fertility, a multi-functional plasmid vector was introduced into the bacterium Pseudomonas aeruginosa containing the Dichelobacter nodosus fimbrial subunit gene with a grafted amino acid sequence of luteinizing hormone releasing hormone (LHRH) peptide. Fimbriae of this recombinant, when run on a SDC-polyacrylamide electrophoresis gel, gave a single broad band for LHRH peptide/ D. nodosus subunit and were harvested to produce the anti-fertility vaccine which, when injected into mice, produced atrophy of the testes with absence of sperm, resulting in reversible castration. Double immunogold labelling of the recombinant P. aeruginosa bacteria demonstrated fimbriae with strong expression of the LHRH-peptide, expression of the D. nodosus subunit and absence of host fimbriae.