Prostaglandin E2 induces transcription of skeletal muscle mass regulators interleukin-6 and muscle RING finger-1 in humans.

Cyclooxygenase (COX) inhibiting drugs augment muscle mass and strength improvements during resistance exercise based treatment of sarcopenia in older individuals. Initial evidence suggests a potential mechanism of COX inhibitor blunted prostaglandin (PG) E2 stimulation of interleukin (IL)-6 and the ubiquitin ligase MuRF-1, reducing their inhibition on muscle growth. The purpose of this investigation was to determine if PGE2 stimulates IL-6 and MuRF-1 transcription in skeletal muscle. Muscle biopsies were obtained from 10 young individuals and incubated ex vivo with PGE2 or control and analyzed for IL-6 and MuRF-1 mRNA levels. PGE2 upregulated (P<0.05) expression of both IL-6 (195%) and MuRF-1 (51%). A significant relationship was found between IL-6 and MuRF-1 expression after incubation with PGE2 (r=0.77, P<0.05), suggesting regulation through a common pathway. PGE2 induces IL-6 and MuRF-1 transcription in human skeletal muscle, providing a mechanistic link between COX inhibiting drugs, PGE2, and the regulation of muscle mass.

Resistance exercise and cyclooxygenase (COX) expression in human skeletal muscle: implications for COX-inhibiting drugs and protein synthesis.

We have shown that ibuprofen and acetaminophen block cyclooxygenase (COX) synthesis of prostaglandin PGF(2alpha) and the muscle protein synthesis increase following resistance exercise. Confusingly, these two drugs are purported to work through different mechanisms, with acetaminophen apparently unable to block COX and ibuprofen able to nonspecifically block COX-1 and COX-2. A recently discovered intron-retaining COX, now known to have three variants, has been shown to be sensitive to both drugs. We measured the expression patterns and levels of the intron 1-retaining COX-1 variants (-1b1, -1b2, and -1b3), COX-1, and COX-2 at rest and following resistance exercise to help elucidate the COX through which PGF(2alpha), ibuprofen, and acetaminophen regulate muscle protein synthesis. Skeletal muscle biopsy samples were taken from 16 individuals (8M, 8F) before, 4, and 24 h after a bout of resistance exercise and analyzed using real-time RT-PCR. Relatively few individuals expressed the intron 1-retaining COX-1b variants (COX-1b1, -1b2, and -1b3) at any time point, and when expressed, these variants were in very low abundance. COX-1 was the most abundant COX mRNA before exercise and remained unchanged (P > 0.05) following exercise. COX-2 was not expressed before exercise, but increased significantly (P < 0.05) at 4 and 24 h after exercise. The inconsistent and low levels of expression of the intron 1-retaining COX-1 variants suggest that these variants are not likely responsible for the inhibition of PGF(2alpha) production and skeletal muscle protein synthesis after resistance exercise by ibuprofen and acetaminophen. Skeletal muscle-specific inhibition of COX-1 or COX-2 by these drugs should be considered.

A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice.

BACKGROUND: Olfactorily mediated puberty acceleration in female mice (measured by an increase in uterine weight) has been observed since the 1960s without the active chemosignal being structurally identified. There are many controversies in the literature as to whether this male-originated pheromone is a volatile substance. We investigated the chemical nature of the urinary fractions that are responsible for the characteristic uterine weight increases. RESULTS: The active pheromone was identified as 5,5-dimethyl-2-ethyltetrahydrofuran-2-ol and/or its open-chain tautomer (6-hydroxy-6-methyl-3-heptanone). A series of cyclic vinyl ethers were isolated from chromatographically active fractions of the urine. Because these compounds did not accelerate puberty, we postulated that these ethers were degradation products of a lactol (5,5-dimethyl-2-ethyltetrahydrofuran-2-ol). The lactol was then detected directly in the mouse urine extract using a silylation agent. Synthetic 6-hydroxy-6-methyl-3-heptanone had strong biological activity, whereas its close structural analogs did not. CONCLUSIONS: The male house mouse excretes into its urine a large quantity of a volatile substance that has a unique lactol/hydroxyketone structure. This substance is capable of binding to the less volatile urinary constituents, such as proteins or peptides, and is active in puberty-acceleration bioassays. The controversies regarding the volatility of the puberty-accelerating pheromones can now be explained by considering a complex of volatile lactol/hydroxyketone and urinary proteins.

Stereoselectivity in mammalian chemical communication: male mouse pheromones.

Two male mouse pheromones, 3,4-dehydro-exo-brevicomin (DHB) and 2-sec-butyldihydrothiazole (SBT), are chiral molecules which were previously tested in their respective bioassays as racemic mixtures. The focus of this study has been to determine the absolute configuration of their natural forms and its relation to stereospecific biological action. DHB was established as the R,R-enantiomer possessing biological activity. Due to an extremely easy racemization of SBT under very mild conditions, enantioselectivity of its transmission and its action at the receptor site appear to be of secondary importance.

Putative chemical signals from white-tailed deer (Odocoileus virginianus). Urinary and vaginal mucus volatiles excreted by females during breeding season.

Urine and vaginal mucus samples from female white-tailed deer in estrus and mid-cycle were analyzed by combined gas chromatography-mass spectrometry (GC-MS). Forty-four volatiles were found in mucus and 63 in urine. The volatiles common to both vaginal mucus and urine included alcohols, aldehydes, furans, ketones, alkanes, and alkenes. Aromatic hydrocarbons were present only in the vaginal mucus, whereas pyrans, amines, esters, and phenols were found only in urine. Both estrous mucus and estrous urine could be identified by the presence of specific compounds not present in mid-cycle samples. Numerous compounds exhibited dependency on ovarian hormones.

Inhibition of sexual maturation in juvenile female and male mice by a chemosignal of female origin.

Maturation of juvenile house mice (Mus domesticus) exposed to synthetic 2,5-dimethylpyrazine was compared to the maturation of juvenile male and female mice exposed to urine from adult males and grouped females. Exposure of juvenile male mice to the synthetic compound significantly inhibited the growth of their testes and sex-accessory glands in a fashion similar to the urine of adult males and grouped females. The uterine weight and the presence of ova in the oviducts in females was strongly affected by synthetic stimuli. The majority of females (76-84%) exposed either to grouped females' urine or to the synthetic compound did not exhibit significant uterine weight increase and did not achieve pubertal ovulation before the age of 30 days as it has been reported for control mice. It is suggested that 2,5-dimethylpyrazine delays puberty of juvenile conspecifics in a population and may function whenever the living conditions of growing animals are below the level required for successful reproduction.

Chemical characterization of urinary volatile compounds ofPeromyscus californicus, a monogamous biparental rodent.

The urinary profiles of adult female and male California mice were examined to determine the volatile compounds that may affect reproduction. The urinary volatiles identified by gas chromatography-mass spectrometry (GC-MS) include ketones, pyrazines, alkanes, nitrile, and aldehyde. None of volatile compounds was specific to males or females. The concentration of urinary volatiles in pregnant and lactating females was significantly higher than in virgin females. Male urinary volatile concentrations were similar to those of pregnant and lactating females. The GC profiles of both sexes were distinguished by a high percentage (36%) of pyrazine derivatives that were also in unusually high concentrations. These compounds may play a wide-ranging role in the control of reproduction in the California mouse.

Long-term effect of a urinary chemosignal on reproductive fitness in female mice.

The effect of synthetic 2,5-dimethylpyrazine (a compound naturally produced by grouped females) on the overall reproductive success of female mice was investigated. The exposure of tested females to control or experimental stimuli began on the day of birth and lasted throughout maturation, mating, pregnancy, and the lactating of their first litters. Females exposed to this urinary compound attained their first estrus significantly later than the animals exposed to water only. On average, the prepubertal, exposed females reached puberty 3.7 to 3.9 days later than the unexposed animals. The females in which puberty was delayed by this synthetic chemosignal did not show a single fully completed estrous cycle before the age of 35 days. Only 52-64% of exposed females successfully bred and reared their litters as compared to 86-96% of the unexposed, control females (p < 0.05). A significantly higher mortality of pups associated with 2,5-dimethylpyrazine exposure during lactation was observed. The reproductive deficit displayed by females born into a 2,5-dimethylpyrazine environment is qualitatively similar to the effect observed in dense populations, in both laboratory and free-living conditions.

Urine marking in male mice: responses to natural and synthetic chemosignals.

The effects of natural chemosignals and their synthetic terpenic analogs on urinary marking in mice were investigated. The number and size of marking spots, and percentage of response and avoidance/counter-marking behavior, were measured for dominant and subordinate males. The males' marking behavior was found to differ significantly based on the type of stimulus sample used. Marking behavior in response to female urine was significantly altered by addition of synthetic analogs (farnesene isomers) to it. Both dominant and subordinate males avoided marking the section of the test area soiled with stimulus samples containing either natural or synthetic farnesenes. Differential responses of the dominant and subordinate male mice were observed.

Socio-sexual olfactory preference in female mice: attractiveness of synthetic chemosignals.

Two sesquiterpenic compounds, E,E,-alpha-farnesene and E-beta-farnesene, which were previously found as major constituents of the male mouse preputial glands, were tested for their attractiveness to female mice. Sexually naive and sexually experienced females were given the opportunity to choose between natural stimuli and synthetic analogs of preputial chemosignals. Naive females preferred investigating the odors of intact males' urine and synthetic farnesenes when spiked in high concentration in bladder urine or water over control stimulus (water or bladder urine alone). Investigatory preference was not observed when synthetic farnesenes were presented to naive females in low concentration, i.e., only twice the natural content in the dominant male urine. However, sexually experienced females were clearly able to recognize and prefer samples with synthetic farnesenes, even in low concentration. We suggest that those sesquiterpenic compounds may play a wide-ranging role in the female recognition of sexually mature and socially dominant males.

Thet complex of the mouse: Chemical characterization by urinary volatile profiles.

Urine samples from C3H congenic house mice (Mus domesticus) differing only at thet complex were examined by capillary gas chromatography to assess variations in the volatile components that may cause olfactory discrimination between animals bearingt lethal and+(wild-type) haplotypes. Urine was collected from 192 males and females varying in age from 1 to 9 months. C3H congenic mice that have the same genetic background at all loci but differed in theirt complex genotypes: +/ +, +/tw1,T/t w1, T/+ were used. No urinary volatiles were unique to thet complex. However, significant differences amongt complex genotypes and among ages occurred for concentrations of 12 male volatiles and four female volatiles. Usually young males (1-2 months of age) had significantly higher concentrations of cyclic enol ethers and ketones than older males (4-9 months of age). Moreover, some urinary volatiles (cyclic enol ethers, one ketone, dehydrobrevicomin, and thiazoline) were excreted in the urine of T/+and/orT/t males in significantly higher concentration than in the urine of +/+ males. Age andt complex genotype influences on the urinary volatiles in females were observed for four ketones. Gas chromatography of urinary components has the potential to be used in field studies of thet complex because the two t complex genotypes found in wild populations, +/+ and +/t, had significant differences in concentration for two males volatiles and three female volatiles.

Chemistry of male dominance in the house mouse, Mus domesticus.

Two terpenic constituents, E,E,-alpha-farnesene and E-beta-farnesene, were found to be elevated in dominant male urine when compared to subordinate or control males. These two urinary compounds were absent in the bladder urine of males; however, they were the most prominent constituents of the perputial gland's aliquots. The results of a two-choice preference test, conducted on ICR/Alb subordinate males, gave a strong indication that these two terpenic constituents introduced into the previously attractive stimulus significantly discouraged prolonged investigations by male mice. The compounds, whether present in the urine matrix or water, rendered the stimulus with a quality behaviorally similar to the urine of dominant males. It appears that they may be synonymous with the previously described aversion signal produced by dominant males. We suggest that these compounds may play a wide-ranging role in the territorial marking behavior of male mice.

Puberty-affecting synthetic analogs of urinary chemosignals in the house mouse, Mus domesticus.

Endocrinologically- and socially-dependent volatile constituents of female mouse urine, identified in a previous study, were tested for their capability to accelerate puberty and extend the estrous period in young females. Several volatile ketones advanced puberty by approximately three days and extended the period of vaginal cornification in 55-75% of exposed females. High High concentrations of these substances were capable of overriding the known puberty-delaying chemosignals. Volatile cyclic enol ethers were also effective in extending estrus, but not puberty acceleration.

Urinary volatile profiles of pine vole,Microtus pinetorum, and their endocrine dependency.

The volatile compounds identified by combined gas chromatography-mass spectrometry inMicrotus pinetorum urine include alcohols, aldehydes, hydrocarbons, ketones, nitriles, and pyrazines. Several lactone derivatives were found to be characteristic urinary substances of this species. Ovariectomy depressed concentrations of only five out of a great number of profile constituents. Elevating estrogen levels (by exposing females to male-soiled bedding or treating them with estradiol) tends to depress the urinary concentration of a number of selected volatiles. Estrogen implantation provoked a periodic increase in the level of three compounds (nonanal, benzal-dehyde, and an unidentified substance). The volatile profile of castrate male urine was similar to that of intact male urine. Female urine contained γ-octanoic lactone and two pyrazine derivatives in higher concentrations andp-methyI-propenylbenzene in a lower concentration, when compared to male urine. No qualitative differences between the urinary profiles of males and females were observed.

Pattern of volatile compounds in dominant and subordinate male mouse urine.

The urinary volatiles from dominant and subordinate male mice were chromatographically compared, both immediately and seven days after dominant-subordinate relationships between pairs were formed. Statistical comparison of the peak areas of volatile constituents present in male urine revealed that 16 urinary compounds exhibited substantial concentration differences depending upon social status of the animals. Urinary dihydrofurans, ketones, and acetates exhibited a significant, long-term (seven-day) decrease in the urine of subordinates when compared to control and dominant males. Two sesquiterpenic compounds, α- and ß-farnesene, were elevated only in the dominant urine one week after dominance was established. 2-(sec-Butyl)-4,5-dihydrothiazole was found in higher concentration in bladder or excreted urine of dominant males when compared to subordinates. Of the 16 compounds subjected to statistical analysis, four exhibited hormonal dependency: α- and ß-farnesene, dehydro-exo-brevicomin, and 2-(sec-butyl)-4,5-dihydrothiazole. Neither dehydro-exo-brevicomin nor 2-(sec-butyl)-4,5-dihydrothiazole was present in the urine of immature and castrated males. Testosterone treatment restores their presence in male urine. Also, α- and ß-farnesene were absent in the urine of immature males and significantly reduced in the urine of castrated males. The absence of α- and ß-farnesene in bladder urine suggests that one of the sex-accessory glands may be the site of their origin.

Dynamics of excretion of urinary chemosignals in the house mouse (Mus musculus) during the natural estrous cycle.

The volatile fraction of urinary metabolites was investigated chromatographically at five different stages of the natural estrous cycle. A very substantial endocrine dependency has been noted for 11 compounds: 4 ketones, 2 acetate esters, 3 dihydrofuran isomers, dehydro-exo-brevicomin, and 2,5-dimethylpyrazine. The compounds were structurally verified through combined gas chromatography/mass spectrometry.

Variations in mouse (Mus musculus) urinary volatiles during different periods of pregnancy and lactation.

Mouse urine samples from different pregnancy and lactation periods were examined by capillary gas chromatography to assess variations in the volatile signals that may affect the endocrine function of other females. Statistically significant changes in the excretion of certain urinary volatiles were observed; from 26 readily quantifiable constituents, 14 appear to be under the endocrine control. These selected components, positively identified through mass spectrometry and retention data, and the synthetic standards are ketones, unsaturated alcohols, esters, and cyclic vinyl ethers.

Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents.

Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-brevicomin, were synthesized and tested for their ability to induce estrous cycle in female mice (the Whitten effect). The suppression of ovarian cycling activity that resulted from grouping the females was abolished by exposure to normal male urine. The synthetic compounds, when added together in appropriate concentrations to the (previously inactive) urine of castrated males, or even to water, were found to be as effective as normal male urine. The action of the synthetic compounds appears attenuated for singly caged females.

Adrenal-mediated endogenous metabolites inhibit puberty in female mice.

While assessing a potential role of adrenal glands in the production of the hitherto unidentified puberty-delaying pheromone of female mice, the urinary volatile profiles of normal and adrenalectomized animals were quantitatively compared. Six components, whose concentrations were depressed after adrenalectomy, were identified: 2-heptanone, trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate, cis-2-penten-1-yl acetate, and 2,5-dimethylpyrazine. When these laboratory-synthesized chemicals were added (in their natural concentrations) to either previously inactive urine from adrenalectomized females or plain water, the biological activity was fully restored.

Synthetic pheromones that promote inter-male aggression in mice.

Two volatile constituents of male mouse urine, dehydro-exo-brevicomin and 2-(sec-butyl)-dihydrothiazole, have been found active in bioassays of inter-male aggressive behavior. The two synthetic compounds act synergistically when added to castrated male urine but not when added to water, and they provoke fighting that is quantitatively and qualitatively comparable to that elicited by intact male urine.