Direct comparison of the effects of intravenous kisspeptin-10, kisspeptin-54 and GnRH on gonadotrophin secretion in healthy men.

STUDY QUESTION: How potently does the novel hypothalamic stimulator of reproduction, kisspeptin, increase gonadotrophin secretion when compared with GnRH in healthy men? SUMMARY ANSWER: At the doses tested, intravenous administration of either of two major kisspeptin isoforms, kisspeptin-10 and -54, was associated with similar levels of gonadotrophin secretion in healthy men; however, GnRH was more potent when compared with either kisspeptin isoform. WHAT IS KNOWN ALREADY: Kisspeptin-10 and -54 are naturally occurring hormones in the kisspeptin peptide family which potently stimulates endogenous GnRH secretion from the hypothalamus, so have the potential to treat patients with reproductive disorders. Rodent studies suggest that kisspeptin-54 is more potent when compared with kisspepitn-10; however, their effects have not previously been directly compared in humans, or compared with direct pituitary stimulation of gonadotrophin secretion using GnRH. STUDY DESIGN, SIZE AND DURATION: A single-blinded placebo controlled physiological study was performed from January to December 2013. Local ethical approval was granted, and five participants were recruited to each dosing group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthy men were administered vehicle, kisspeptin-10, kisspeptin-54 and GnRH intravenously for 3 h on different study days. Each hormone was administered at 0.1, 0.3 and 1.0 nmol/kg/h doses (n = 5 subjects per group). Regular blood sampling was conducted throughout the study to measure LH and FSH. Study visits were conducted at least a week apart. MAIN RESULTS AND THE ROLE OF CHANCE: Serum LH and FSH levels were ∼3-fold higher during GnRH infusion when compared with kisspeptin-10 and ∼2-fold higher when compared with kisspeptin-54 [mean area under the curve serum LH during infusion (in hours times international units per litre, h.IU/l): 10.81 ± 1.73, 1.0 nmol/kg/h kisspeptin-10; 14.43 ± 1.27, 1.0 nmol/kg/h kisspeptin-54; 34.06 ± 5.18, 1.0 nmol/kg/h GnRH, P < 0.001 versus kisspeptin-10, P < 0.01 versus kisspeptin-54]. LIMITATIONS, REASONS FOR CAUTION: This study had a small sample size. WIDER IMPLICATIONS OF THE FINDINGS: Kisspeptin offers a novel means of stimulating the reproductive axis. Our data suggest that kisspeptin stimulates gonadotrophin secretion less potently when compared with GnRH; however, kisspeptin may stimulate gonadotrophins in a more physiological manner when compared with current therapies. Kisspeptin is emerging as a future therapeutic agent, so it is important to establish which kisspeptin hormones could be used to treat patients with infertility. Results of this study suggest that either isoform has similar effects on reproductive hormone secretion in healthy men when administered intravenously. STUDY FUNDING/COMPETING INTERESTS: This work is funded by grants from the MRC and NIHR and is supported by the NIHR Imperial Biomedical Research Centre Funding Scheme. C.N.J. is supported by an NIHR Clinical Lectureship. A.A. is supported by Wellcome Trust Research Training Fellowships. A.N.C. is supported by Wellcome Trust Translational Medicine Training Fellowship. W.S.D. is supported by an NIHR Career Development Fellowship.

Twice-daily subcutaneous injection of kisspeptin-54 does not abolish menstrual cyclicity in healthy female volunteers.

BACKGROUND: Kisspeptin is a critical hypothalamic regulator of reproductive function. Chronic kisspeptin administration causes profound tachyphylaxis in male monkeys and in women with functional hypothalamic amenorrhea. The pharmacological effects of chronic kisspeptin exposure in healthy women with normal menstrual cycles have not been studied previously. AIM: Our aim was to determine the effects of follicular-phase kisspeptin-54 treatment on menstrual cyclicity in healthy women. METHODS: We performed a prospective, single-blinded, 1-way crossover study. Healthy women received twice-daily sc injections of kisspeptin (6.4 nmol/kg) or 0.9% saline during menstrual days 7-14 (n = 5 per treatment arm). Serial assessments of basal reproductive hormones, ultrasound parameters, LH pulsatility, and acute sensitivity to GnRH and kisspeptin-54 injection were performed. RESULTS: Menstrual cyclicity persisted in all women after follicular-phase kisspeptin-54 treatment. Chronic exposure to kisspeptin-54 did not abolish acute stimulation of LH after injection of kisspeptin-54 or GnRH. In addition, kisspeptin-54 treatment was associated with a shorter mean length of the menstrual cycle (mean length of menstrual cycle was 28.6 ± 1.4 days with saline vs 26.8 ± 3.1 days with kisspeptin, P < .01), earlier onset of highest recorded serum LH (mean menstrual day of highest LH was 15.2 ± 1.3 with saline vs 13.0 ± 1.9 with kisspeptin, P < .05), and earlier onset of the luteal phase (mean menstrual day of progesterone increase was 18.0 ± 2.1 with saline vs 15.8 ± 0.9 with kisspeptin, P < .05). CONCLUSION: Our data suggest that 1 week of exogenous kisspeptin-54 does not abolish menstrual cyclicity in healthy women. Further work is needed to determine whether kisspeptin could be used to treat certain anovulatory disorders.

Modulation of the hepatic expression of the estrogen-regulated mRNA stabilizing factor by estrogenic and antiestrogenic nonsteroidal xenobiotics.

Estrogen-mediated accumulation of apolipoprotein II (apoII) mRNA in the avian liver is due, in part, to its stabilization. This stabilization appears to be due to the estrogen-regulated mRNA stabilizing factor (E-RmRNASF) that is expressed in response to estrogen. The E-RmRNASF protects the mRNA from targeted endonucleolytic degradation (Ratnasabapathy, Cell Mol Biol Res 41: 583-594, 1995). To determine whether certain environmental xenobiotics altered the expression of the gene encoding E-RmRNASF by mimicking estrogen, roosters were given estrogen, tamoxifen, clomiphene, hexachlorophene, lindane, rotenone, chlordecone, dichlorodiphenyltrichloroethane (DDT); Araclor, methoxychlor, dieldrin, toxaphene, or bisphenol-A parenterally. Uniformly radiolabeled, capped, and polyadenylated apoII mRNA, incubated in vitro in the presence of liver cytosolic extracts from birds that received estrogen, tamoxifen, hexachlorophene, chlordecone, or Araclor, remained stable, indicating that these agents were estrogenic and stimulated the expression of E-RmRNASF. However, the same mRNA was degraded in similar extracts from control roosters and those treated with clomiphene, DDT, methoxychlor, dieldrin, rotenone, toxaphene, lindane, or bisphenol-A. To determine whether the latter agents were antiestrogenic, roosters were given a 1:5 molar combination of estrogen and each of the xenobiotics. ApoII mRNA showed degradation in liver extracts from roosters that received clomiphene, toxaphene, or bisphenol-A, indicating that these agents prevented estrogenic stimulation of expression of the E-RmRNASF and were antiestrogenic. However, the rest of the xenobiotics failed to antagonize estrogenic stimulation of E-RmRNASF gene expression. These results set a precedent in showing the estrogenic and antiestrogenic effects in vivo of environmental xenobiotics on the expression of a regulatory protein involved in estrogen-mediated mRNA stabilization.

In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver.

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.

Characterization of the inducer of short transcripts, a human immunodeficiency virus type 1 transcriptional element that activates the synthesis of short RNAs.

The inducer of short transcripts, or IST, is an unusual transcriptional element located downstream of the human immunodeficiency virus type 1 (HIV-1) promoter. IST activates HIV-1 transcription, but the resulting RNAs are short and end at approximately position +59. IST, therefore, appears to promote the formation of transcription complexes that are unable to elongate efficiently. This activity contrasts with that of TAR, the target for Tat trans-activation, which upon binding of the viral protein Tat promotes the formation of transcription complexes capable of efficient elongation through the entire viral genome. We have localized and characterized the IST element. Our results indicate that IST is located mainly between positions -5 and +26, although the sequences from positions +40 to +59 also contribute to IST activity. Unlike TAR, which is an RNA element, IST appears to be a DNA element. Thus, the HIV-1 R region is a complex regulatory region with RNA and DNA elements that promote the formation of transcription complexes with different elongation properties.

The HIV-1 long terminal repeat contains an unusual element that induces the synthesis of short RNAs from various mRNA and snRNA promoters.

We describe an unusual element that activates the synthesis of short transcripts from a wide variety of mRNA and small nuclear RNA (snRNA) promoters, including the U6 RNA polymerase III promoter. This inducer of short transcripts (IST) is located between positions -5 and +82 relative to the cap site in the HIV-1 LTR. In the presence of IST, the total transcriptional activity of the different promoters is greatly increased, but the resulting additional RNA molecules are short, ending around position +60. IST is not the RNA target (TAR) for Tat trans-activation; however, because it relies entirely on cellular factors for activity, IST may serve to provide abundant RNA targets for Tat trans-activation without a requirement for full-length viral mRNA expression.

The 3'-untranslated region of apolipoprotein II mRNA contains two independent domains that bind distinct cytosolic factors.

The 3'-untranslated region of apolipoprotein II (apoII) mRNA contains target sites for mRNA breakdown (Binder, R., Hwang, S.-P. L., Ratnasabapathy, R., and Williams, D. L. (1989) J. Biol. Chem. 264, 16910-16918). Degradation occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-untranslated region. Degradation target sites occur in two clusters that are localized within two larger domains of secondary structure. In this study, gel shift and label transfer assays were used to identify liver cytosolic factors that recognize the 3'-untranslated region of apoII mRNA. The results show preferential binding of cytosolic factors to the 3'-untranslated region as compared to the coding region. UV cross-linking experiments confirmed that cytosolic factors labeled by the 3'-untranslated region are a subset of proteins labeled by the entire mRNA. Two distinct binding domains were identified within the 3'-untranslated region. The upstream domain encompassing nucleotides 400-547 extends from the translation stop codon through the complex stem-loop D structure described previously. This domain labeled primarily a 34-kDa protein in UV cross-linking experiments. The downstream binding domain encompassing nucleotides 568-643 includes another region of secondary structure and terminates within the universal polyadenylation signal. The downstream domain labeled primarily a 60-kDa protein in UV cross-linking experiments. The upstream and downstream binding domains did not compete with each other in gel shift or cross-linking experiments. These results indicate that the 3'-untranslated region can form two independent messenger ribonucleoprotein complexes localized to domains that include target sites for apoII mRNA degradation. We speculate that these messenger ribonucleoprotein complexes may play a role in the degradation of apoII mRNA or in the regulation of this process.

Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region.

Degradation intermediates of the estrogen-regulated apolipoprotein (apo) II mRNA were identified by S1 nuclease mapping and primer extension analysis. S1 mapping of poly(A)-RNA detected a series of mRNAs truncated at specific sites in the 3'-noncoding region. Many of these sites were also detected by primer extension analysis indicating that truncated molecules resulted from endonucleolytic cleavage in the 3'-noncoding region. Identical cleavage sites were seen with RNA from estrogen-treated animals or from animals withdrawn from hormone under conditions where apoII mRNA degraded in the slow (t1/2 = 13 h) or rapid (t1/2 = 1.5 h) decay mode. No differences were seen in poly(A) tail length or heterogeneity among these conditions. These results indicate that the estrogen-induced alteration in apoII mRNA turnover does not involve a new pathway of degradation, but, more likely, involves an increased targeting of the mRNA for degradation by a preexisting pathway. These data are consistent with a mechanism in which the initial step in apoII mRNA degradation is an endonucleolytic cleavage in the 3'-noncoding region without prior removal of the poly(A) tail. The endonucleolytic cleavage sites occurred predominantly at 5'-AAU-3' or 5'-UAA-3' trinucleotides found in single-stranded domains in a secondary structure model of the naked mRNA (Hwang, S-P. L., Eisenberg, M., Binder, R., Shelness, G. S., and Williams, D. L. (1989) J. Biol. Chem. 264, 8410-8418). The structure of the 3'-noncoding region in polyribosomal messenger ribonucleoprotein was examined by titrations of liver homogenates with dimethyl sulfate and cobra venom RNase. The results suggest that the typical cleavage site is a 5'-AAU-3' or 5'-UAA-3' trinucleotide in an accessible single-stranded loop domain. Single-stranded domains alone or accessible domains alone are not sufficient for cleavage. Similarly, 5'-AAU-3' or 5'-UAA-3' trinucleotides alone are not sufficient for cleavage. Localization of these trinucleotides to accessible single-stranded domains in the polyribosomal messenger ribonucleoprotein may provide the specificity for cleavage during targeted degradation.