Characterization of the inhibitory roles of RFRP3, the mammalian ortholog of GnIH, in the control of gonadotropin secretion in the rat: in vivo and in vitro studies.

RF-amide related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone (GnIH), have been proposed as key regulators of gonadotropin secretion in higher vertebrates. Yet considerable debate has arisen recently on their physiological relevance and potential mechanisms and sites of action. Present studies were undertaken to further characterize the effects of RFRP on LH and FSH secretion by a combination of in vivo and in vitro approaches in male and female rats. Initial screening via intracerebroventricular (icv) administration of different analogs of RFRP1 (RFRP1-12 and RFRP1-20) and RFRP3 (RFRP3-8 and RFRP3-17), as well as the related neuropeptide FF (NPFF8), to gonadectomized (GNX) female rats evidenced significant, albeit modest, inhibitory effects on LH secretion only for RFRP3-8 and RFRP3-17, which were detectable at the high dose rage (1 nmol for RFRP3-8, 5 nmol for RFRP3-17). This moderate inhibitory action was also documented after icv administration of RFRP3-8 to intact and GNX male rats. In addition, systemic (intravenous) administration of RFRP3-8 decreased the circulating levels of both gonadotropins in GNX male rats. Likewise, RFRP3-8 inhibited basal and GnRH-stimulated LH secretion by pituitaries from GNX males in vitro. This inhibitory effect was blocked by the antagonist of RFRP receptors, RF9. In summary, our results support a putative inhibitory role of RFRP3 as ortholog of GnIH in the regulation of gonadotropin secretion in mammals, which appears to involve direct pituitary actions as well as potential central (hypothalamic) effects.

Characterization of the potent gonadotropin-releasing activity of RF9, a selective antagonist of RF-amide-related peptides and neuropeptide FF receptors: physiological and pharmacological implications.

Identification of RF-amide-related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone, has drawn considerable interest on its potential effects and mechanisms of action in the control of gonadotropin secretion in higher vertebrates. Yet, these analyses have so far relied mostly on indirect approaches, while direct assessment of their physiological roles has been hampered by the lack of suitable antagonists. RF9 was recently reported as a selective and potent antagonist of the receptors for RFRP (RFRPR) and the related neuropeptides, neuropeptide FF (NPFF) and neuropeptide AF (NPFF receptor). We show here that RF9 possesses very strong gonadotropin-releasing activities in vivo. Central administration of RF9 evoked a dose-dependent increase of LH and FSH levels in adult male and female rats. Similarly, male and female mice responded to intracerebroventricular injection of RF9 with robust LH secretory bursts. In rats, administration of RF9 further augmented the gonadotropin-releasing effects of kisspeptin, and its stimulatory effects were detected despite the prevailing suppression of gonadotropin secretion by testosterone or estradiol. In fact, blockade of estrogen receptor-alpha partially attenuated gonadotropin responses to RF9. Finally, systemic administration of RF9 modestly stimulated LH secretion in vivo, although no direct effects in terms of gonadotropin secretion were detected at the pituitary in vitro. Altogether, these data are the first to disclose the potent gonadotropin-releasing activity of RF9, a selective antagonist of RFRP (and NPFF) receptors. Our findings support a putative role of the RFRP/gonadotropin-inhibitory hormone system in the central control of gonadotropin secretion in mammals and have interesting implications concerning the potential therapeutic indications and pharmacological effects of RF9.

Follicle-stimulating hormone responses to kisspeptin in the female rat at the preovulatory period: modulation by estrogen and progesterone receptors.

Ovulation is triggered by the preovulatory surge of gonadotropins that, in rodents, is defined by the concomitant rise in circulating LH and FSH at the afternoon of proestrus (primary surge), followed by persistently elevated FSH levels at early estrus (secondary surge). In recent years, kisspeptins, products of the KiSS-1 gene that act via G protein-coupled receptor 54, have emerged as an essential hypothalamic conduit for the generation of the preovulatory LH surge by conveying positive feedback effects of estradiol onto GnRH neurons, an event that involves not only estradiol-induced transcription of the KiSS-1 gene at the anteroventral periventricular nucleus but also its ability to modulate GnRH/LH responses to kisspeptin. However, little is known about the potential modulation of FSH responsiveness to kisspeptin by sex steroids in the cyclic female. We report herein analyses on the consequences of selective blockade of estrogen receptors (ER)-alpha and -beta, as well as progesterone receptor (PR), on the ovulatory surges of FSH and their modulation by kisspeptin. Antagonism of ERalpha or PR equally blunted the primary and secondary surges of FSH and nullified FSH responses to kisspeptin at the preovulatory period. Conversely, selective blockade of ERbeta failed to induce major changes in terms of endogenous FSH surges, yet it decreased FSH responses to exogenous kisspeptin. In contrast, FSH responses to GnRH were fully conserved after ERbeta blockade and partially preserved after inhibition of ERalpha and PR signaling. Finally, secondary FSH secretion was rescued by kisspeptin in females with selective blockade of ERalpha but not PR. In sum, our results substantiate a concurrent, indispensable role of ERalpha and PR in the generation of FSH surges and the stimulation of FSH responses to kisspeptin at the ovulatory period. In addition, our data suggest that ERbeta might operate as a subtle, positive modulator of the preovulatory FSH responses to kisspeptin, a role that is opposite to its putative inhibitory action on kisspeptin-induced LH secretion and might contribute to the dissociation of gonadotropin secretion at the ovulatory phase in the cyclic female rat.

Opposite roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of luteinizing hormone responses to kisspeptin in the female rat: implications for the generation of the preovulatory surge.

Ovulation is triggered by the preovulatory rise of gonadotropins, which is in turn elicited by the preceding increase in circulating estrogen. Kisspeptins, ligands of G protein-coupled receptor 54 encoded by the KiSS-1 gene, have emerged as potent stimulators of GnRH/LH secretion, and KiSS-1 neurons at the anteroventral periventricular nucleus have been shown to be involved in the generation of preovulatory LH surge, estrogen being a potent elicitor of KiSS-1 gene expression selectively at the anteroventral periventricular nucleus. Whether, in addition to transcriptional effects, estrogen influences other aspects of kisspeptin-induced GnRH/LH release in the female remains unexplored. We provide herein evidence for the specific roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of LH responses to kisspeptin and the generation of the preovulatory surge. Selective blockade of ERalpha in cyclic females blunted LH responses to kisspeptin, eliminated the endogenous preovulatory rise of LH, and blocked ovulation. In contrast, antagonism of ERbeta failed to cause major changes in terms of LH surge and ovulatory rate but significantly augmented acute LH responses to kisspeptin. Notably, defective LH secretion and ovulation after ERalpha blockade were not observed after GnRH stimulation, which elicited maximal acute (<2 h) LH responses regardless of ERalpha/ERbeta signaling. In addition, net LH secretion in response to kisspeptin was decreased by ovariectomy and increased after selective activation of ERalpha but not ERbeta. Altogether, our data document the prominent positive role of ERalpha in the regulation of GnRH/LH responsiveness to kisspeptin and, thereby, ovulation. In addition, our results disclose the putative function of ERbeta as negative modifier of GnRH/LH response to kisspeptin, a phenomenon that might contribute to partially restraining LH secretion at certain physiological states.

Redox effects on the excited-state lifetime in chlorosomes and bacteriochlorophyll c oligomers.

Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively.

Self quenching of chlorosome chlorophylls in water and hexanol-saturated water.

The optical properties of a methyl ester homolog of bacteriochlorophylld (BChld M ) and bacteriochlorophyllc (BChlc) in H2O, hexanol-saturated H2O and methanol were studied by absorption, fluorescence emission, and circular dichroism (CD). In H2O, BChld M spontaneously forms an aggregate similar to that formed in hexane, with absorption maximum at 730 nm and fluorescence emission at 748 nm. For the pigment sample in hexanol-saturated H2O, while the absorption peaks at 661 nm, only slightly red-shifted compared to the monomer, the fluorescence emission is highly quenched. When diluted 2-3 fold with H2O, the absorption returns to around 720 nm, characteristic of an aggregate. The CD spectrum of the H2O aggregate exhibits a derivative-shaped feature with positive and negative peaks, while the amplitude is lower than that of chlorosomes. The Fourier transform infrared spectra of BChld M aggregates in H2O and hexane were measured. A 1644 cm(-1) band, indicative of a bonded 13(1)-keto group, is detected for both samples. A marker band for 5-coordinated Mg was observed at 1611 cm(-1) for the two samples as well. To study the kinetic behavior of the samples, both single-photon counting (SPC) fluorescence and transient absorption difference spectroscopic measurements were performed. For BChld M in hexanol-saturated H2O, a fast decay component with a lifetime of 10 to 14 ps was detected using the two different techniques. The fast decay could be explained by the concentration quenching phenomenon due to a high local pigment concentration. For the pigment sample in H2O, SPC gave a 16 ps component, whereas global analysis of transient absorption data generated two fast components: 3.5 and 26 ps. The difference may arise from the different excitation intensities. With a much higher excitation in the latter measurements, other quenching processes, e.g. annihilation, might be introduced, giving the 3.5 ps component. Finally, atomic force microscopy was used to examine the ultrastructure of BChld M in H2O and hexanol-saturated H2O. Pigment clusters with diameters ranging from 15 to 45 nm were observed in both samples.

Femtosecond probe of structural analogies between chlorosomes and bacteriochlorophyll c aggregates.

Bacteriochlorophyll c pigments extracted from light harvesting chlorosomes in green photosynthetic bacteria are known to self-assemble into aggregates whose electronic spectroscopy resembles that of intact chlorosomes. Femtosecond optical experiments reveal that the chlorosomes and their reconstituted aggregates exhibit closely analogous internal energy transfer kinetics and exciton state evolution. These comparisons furnish compelling new evidence that proteins do not exert a major local role in the BChl c antenna pigment organization of intact chlorosomes.

Ultrafast energy transfer in light-harvesting chlorosomes from the green sulfur bacterium Chlorobium tepidum.

Two independent pump-probe techniques were used to study the antenna energy transfer kinetics of intact chlorosomes from the green sulfur bacterium Chlorobium tepidum with femtosecond resolution. The isotropic kinetics revealed by one-color experiments in the BChl c antenna were inhomogeneous with respect to wavelength. Multiexponential analyses of the photobleaching/stimulated emission (PB/SE) decay profiles typically yielded (apart from a approximately 10 fs component that may stem from the initial coherent oscillation) components with lifetimes 1-2 ps and several tens of ps. The largest amplitudes for the latter component occur at 810 nm, the longest wavelength studied. Analyses of most two-color pump-probe profiles with the probe wavelength red-shifted from the pump wavelength yielded no PB/SE rise components. PB/SE components with approximately 1 ps risetime were found in 790 --> 810 and 790 --> 820 nm profiles, in which the probe wavelength is situated well into the BChl a absorption region. A 760 --> 740 nm uphill two-color experiment yielded a PB/SE component with 4-6 ps risetime. Broadband absorption difference spectra of chlorosomes excited at 720 nm (in the blue edge of the 746 nm BChl c Qy band) exhibit approximately 15 nm red-shifting of the PB/SE peak wavelength during the first several hundred fs. Analogous spectra excited at 760 nm (at the red edge) show little dynamic spectral shifting. Our results suggest that inhomogeneous broadening and spectral equilibration play a larger role in the early BChl c antenna kinetics in chlorosomes from C. tepidum than in those from C. aurantiacus, a system studied previously. As in C. aurantiacus, the initial one-color anisotropies r(0) for most BChl c wavelengths are close to 0.4. The corresponding residual anisotropies r(infinity) are typically 0.19-0.25, which is much lower than found in C. aurantiacus (> or = 0.35); the transition moment organization is appreciably less collinear in the BChl c antenna of C. tepidum. However, the final one-color anisotropies at 789 and 801 nm are approximately 0 and 0.09 respectively, and the final anisotropy in time 780 --> 800 nm experiment is approximately -0.1. These facts indicate that the BChI a transition moments themselves exhibit some order, and are directed at an angle > 54.7 degrees on the average from the BChl c moments. The one-color profiles exhibit coherent oscillations at most wavelengths, including 800 nm; Fourier analyses of these oscillations frequently yield components with frequencies 70-80 and 130-140 cm-1.

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer.

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Qy absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. 'One-color' and 'two-color' pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides.

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P(*) becomes 4-5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll BA with its environment appears to be altered, possibly because of a change in its position.