Medial Amygdala Kiss1 Neurons Mediate Female Pheromone Stimulation of Luteinizing Hormone in Male Mice.

BACKGROUND/AIMS: The medial amygdala (MeA) responds to olfactory stimuli and alters reproductive physiology. However, the neuronal circuit that relays signals from the MeA to the reproductive axis remains poorly defined. This study aimed to test whether MeA kisspeptin (MeAKiss) neurons in male mice are sensitive to sexually relevant olfactory stimuli and transmit signals to alter reproductive physiology. We also investigated whether MeAKiss neurons have the capacity to elaborate glutamate and GABA neurotransmitters and potentially contribute to reproductive axis regulation. METHODS: Using female urine as a pheromone stimulus, MeAKiss neuronal activity was analysed and serum luteinizing hormone (LH) was measured in male mice. Next, using a chemogenetic approach, MeAKiss neurons were bi-directionally modulated to measure the effect on serum LH and evaluate the activation of the preoptic area. Lastly, using in situ hybridization, we identified the proportion of MeAKiss neurons that express markers for GABAergic (Vgat) and glutamatergic (Vglut2) neurotransmission. RESULTS: Male mice exposed to female urine showed a two-fold increase in the number of c-Fos-positive MeAKiss neurons concomitant with raised LH. Chemogenetic activation of MeAKiss neurons significantly increased LH in the absence of urine exposure, whereas inhibition of MeAKiss neurons did not alter LH. In situ hybridization revealed that MeAKiss neurons are a mixed neuronal population in which 71% express Vgat mRNA, 29% express Vglut2 mRNA, and 6% express both. CONCLUSIONS: Our results uncover, for the first time, that MeAKiss neurons process sexually relevant olfactory signals to influence reproductive hormone levels in male mice, likely through a complex interplay of neuropeptide and neurotransmitter signalling.

Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats.

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar-Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17ß-oestradiol (E2 ). OVX+E2 rats were killed 1 hour after exposure to either: clean bedding, female-soiled bedding or male-soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c-Fos immunohistochemistry revealed that the numbers of Kiss1-expressing cells and c-Fos-immunopositive Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E2 rats exposed to male-soiled bedding than those of the other groups. No significant difference was found with respect to the number of c-Fos-immunopositive Kiss1-expressing cells in the arcuate nucleus and c-Fos-immunopositive Gnrh1-expressing cells between the groups. The number of c-Fos-immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E2 rats exposed to male-soiled bedding than the other groups. OVX+E2 rats exposed to male-soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E2 group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.

Brain processing of a configural vs elemental odor mixture in the newborn rabbit.

Organisms are surrounded throughout life by chemically complex odors. How individuals process an odorant within a mixture or a mixture as a whole is a key question in neuroethology and chemical senses. This question is addressed here by using newborn rabbits, which can be rapidly conditioned to a new stimulus by single association with the mammary pheromone. After conditioning to ethyl maltol (odorant B), pups behaviorally respond to B and an A'B' mixture (68/32 ratio) but not to ethyl isobutyrate (odorant A) or an AB mixture (30/70 ratio). This suggests elemental and configural perception of A'B' and AB, respectively. We then explored the neural substrates underlying the processing of these mixtures with the hypothesis that processing varies according to perception. Pups were pseudoconditioned or conditioned to B on postnatal day 3 before exposure to B, A'B' or AB on day 4. Fos expression was not similar between groups (mainly in the olfactory bulb and posterior piriform cortex) suggesting a differential processing of the stimuli that might reflect either stimulus complexity or conditioning effect. Thus, the ratio of components in A'B' vs AB leads to differential activation of the olfactory system which may contribute to elemental and configural percepts of these mixtures. In addition, together with recent behavioral data, this highlights that configural perception occurs even in relatively immature animals, emphasizing the value of the newborn rabbit for exploration of odor mixture processing from molecules to brain and behavior.

[Chemosignals from isolated females have antimutagenic effect in dividing the cells of bone marrow from male mice of the CBA line].

A level of X-ray induced mitotic disturbances in the cells of the bone marrow of male mice was studied under the modifying influence ofchemosignals from isolated adult female mice of the CBA line. It has been shown that the frequency of chromosomal aberrations in irradiated (4 Gr) males after exposing them for 24 hours on bedding soiled with female chemosignals is lower than in irradiated males in cages with clean bedding. The mechanisms and importance of the antimutagenic effect of female house mouse chemosignals are discussed.

Transfer of [³H]estradiol-17ß and [³H]progesterone from conspecifics to cohabiting female mice.

Estradiol-17ß (E2) and progesterone (P4) play critical roles in female reproductive physiology and behavior. Given the sensitivity of females to exogenous sources of these steroids, we examined the presence of E2 and P4 in conspecifics' excretions and the transfer of excreted steroids between conspecifics. We paired individual adult female mice with a stimulus male or female conspecific given daily injections of [³H]E2 or [³H]P4. Following 48  h of direct interaction with the stimulus animal, we measured radioactivity in the uterus, ovaries, muscle, olfactory bulbs, mesencephalon and diencephalon (MC+DC), and cerebral cortex of the untreated female cohabitant. Radioactivity was significantly present in all tissues of female subjects after individual exposure to a stimulus male or female given [³H]E2. In females exposed to males given [³H]P4, radioactivity was significantly present in the uterus, ovaries, and muscle, but not in other tissues. In females exposed to stimulus females given [³H]P4, radioactivity was significantly present in all tissues except the MC+DC. In mice directly administered [³H]steroids, greater radioactivity was found in the urine of females than of males. Among females directly administered [³H]steroids, greater radioactivity was found in urine of those given [³H]P4 than of those given [³H]E2. When females were administered unlabeled E2 before exposure to [³H]E2-treated females, less radioactivity was detected in most tissues than was detected in the tissues of untreated females exposed to [³H]E2-treated females. We suggest that steroid transfer among individuals has implications for the understanding of various forms of pheromonal activity.

Conception rates to artificial insemination in primiparous, suckled cows exposed to the biostimulatory effect of bulls before and during a gonadotropin-releasing hormone-based estrus synchronization protocol.

The objective of these studies was to evaluate whether exposing primiparous, suckled beef cows to the biostimulatory effect of bulls alters breeding performance associated with an estrus synchronization protocol that included GnRH followed 7 d later by PGF(2alpha) and fixed-time AI (TAI). This was a composite analysis of 3 experiments that evaluated (1) the effects of bull exposure at different days after calving (yr 1); (2) the biostimulatory effects of bull excretory products (yr 2); and (3) the biostimulatory effects of familiar and unfamiliar bulls (yr 3) on the resumption of ovarian cycling activity. In all studies, cows were exposed (biostimulated; n = 94) or not exposed (nonbiostimulated; n = 67) to bulls or excretory products of bulls for at least 60 d before the beginning of the estrus synchronization protocol. Average calving day did not differ among years and was 52 +/- 5 d. Year did not affect the proportions of biostimulated and nonbiostimulated cows that were cycling at the beginning of the estrus synchronization protocol; however, a greater (P < 0.001) proportion of biostimulated than nonbiostimulated cows were cycling at this time. In each year, cows were given GnRH followed by PGF(2alpha) 7 d later. Cows were observed for estrus twice daily (am and pm) after PGF(2alpha). Cows that exhibited estrus before 54, 60, and 64 h after PGF(2alpha) were inseminated by AI 12 h later in yr 1, 2, and 3, respectively. Cows that failed to show estrus were given GnRH and TAI at 62, 72, and 72 h after PGF(2alpha) in yr 1, 2, and 3, respectively. Conception rates were determined by transrectal ultrasonography 35 d after TAI in each year. The percentages of cows that exhibited estrus after PGF(2alpha) and before TAI, the interval from PGF(2alpha) to estrus, and the percentages of cows inseminated 12 h after estrus or at TAI did not differ between biostimulated and nonbiostimulated cows and were 51%, 54.7 +/- 7.3 h, 35%, and 65%, respectively. Conception rates for cows bred by AI 12 h after estrus did not differ between biostimulated and nonbiostimulated cows; however, the TAI conception rate was greater (P < 0.05) for biostimulated cows (57.6%) than for nonbiostimulated cows (35.6%). We conclude that TAI conception rates in an estrus synchronization protocol that includes GnRH followed 7 d later by PGF(2alpha) may be improved by the biostimulatory effect of bulls in postpartum, primiparous cows.

A synthetic fraction of feline facial pheromones calms but does not reduce struggling in cats before venous catheterization.

OBJECTIVE: To evaluate whether a synthetic analogue of feline facial pheromone (FFP) calms cats before, and reduces struggling during intravenous catheterization. DESIGN: Block-randomized, 'blinded' clinical trial. ANIMALS: Seventy-seven healthy cats presented for elective surgery. PROCEDURE: Cats given glycopyrrolate and oxymorphone were assigned to one of four treatments: acepromazine and exposure to FFP (aceFFP); acepromazine and exposure to a placebo (acePlac); exposure to FFP only (FFP) and exposure to placebo only (Plac). The behaviour of cats was recorded on videotape for evaluation by assessors unaware of treatment group. Cats' veins were then catheterized by veterinary students unaware of the study protocol. Based on each cat's response to catheterization, the student independently decided whether intramuscular ketamine was required. RESULTS: Cats in the aceFFP group appeared to be calmer than acePlac cats on the basis of head position and their location in the cage (suggesting benefit from FFP among cats receiving acepromazine) but appeared to be less sedated. Cats in the aceFFP group also appeared to be calmer than FFP cats on the basis of head position and location in the cage. Feline facial pheromone cats were also calmer than Plac cats when compared using body and leg position. Exposure to FFP did not significantly reduce struggling at catheterization, nevertheless, the students were able to catheterize the veins in all cats. CONCLUSION AND CLINICAL RELEVANCE: There were no detrimental behavioural effects associated with either FFP or acepromazine. The FFP had additional calming effects in cats given acepromazine and, to a lesser degree, helped to calm cats that were not given acepromazine. Feline facial pheromone helps to calm cats in unfamiliar surroundings.

The effect of intramuscular injections of boar pheromone 5alpha-androstenol on the hormonal regulation of the estrous cycle in hypoosmatic gilts.

Until 1999 it was accepted that pheromones act exclusively by stimulating the dendritic receptors present in olfactory epithelium. Cycling gilts with an experimentally-disrupted neural olfactory pathway were used to test the hypothesis that boar pheromone 5alpha-androstenol may affect the secretion of hormones involved in the regulation of the estrous cycle by the humoral pathway. On day 12 of the estrous cycle the nasal cavity of gilts (n=15) was irrigated with zink sulfate solution. From day 16 to 20, the experimental group (n=10) was injected intramuscularly with 5alpha-androstenol (20 microg) twice a day. Blood samples were collected from the jugular vein at 4 h intervals on days 17-21 to estimate plasma concentration of LH, oxytocin, estradiol-17beta, testosterone and progesterone. The experimental group displayed a significantly lower mean concentration of LH than the control animals (P<0.0001). The decrease in concentration of LH was accompanied by the reduction of oxytocin (P<0.001), estradiol-17beta (P<0.001) and testosterone (P<0.01) secretion. These results demonstrated that 5alpha-androstenol influenced hormonal regulation by humoral pathway and might be considered to be the priming pheromone in gilts.

A pilot study using synthetic feline facial pheromone for the management of feline idiopathic cystitis.

Synthetic feline facial pheromone (FFP) (Feliway; Ceva Animal Health) was assessed for the management of cats with recurrent feline idiopathic cystitis (FIC). Nine of 12 cats completed the randomised, double-blinded, placebo-controlled, crossover pilot study. They had their environment treated daily with either FFP or placebo for 2 months, after which time the treatment groups were reversed. Owners used visual analogue scales to define the severity of their cat's clinical signs and behavioural changes. Five (56%) of the owners stated that their cat's overall health was better when they were using FFP. Four (44%) of the owners noticed no difference between when using the FFP and when using the placebo. While there were no statistical differences between the two treatment groups there was a trend for the cats exposed to FFP to show fewer days with clinical signs of cystitis (FFP total, mean per cat+/-standard deviation, 30, 4.3+/-6.7; placebo 69, 9.9+/-19.1), a lower overall clinical score (1667, 238+/-476; 2009, 287+/-425), a reduced number of episodes of cystitis (9, 1.3+/-2.0; 10, 1.4+/-2.1) and reduced negative behavioural traits (e.g., less aggression and fear) (-128, -18.3+/-65.8; -73, -10.4+/-35.1).

Experience facilitates vomeronasal and olfactory influence on Fos expression in medial preoptic area during pheromone exposure or mating in male hamsters.

Chemosensory stimuli are essential for mating in male hamsters but either main olfactory or vomeronasal input is sufficient in sexually experienced males. Activation in central chemosensory pathways and medial preoptic area, after stimulation with female chemosignals or after mating, was estimated by counting neurons expressing Fos protein in experienced and naive males, with or without vomeronasal organ lesions. Regions counted included main and accessory olfactory bulbs, corticomedial amygdala, bed nucleus stria terminalis and medial preoptic area. Chemosensory stimulation was more effective in activating medial preoptic area in experienced than in naive males. In experienced males with vomeronasal organs removed, main olfactory input was as effective in activating medial preoptic area as was the combination of main and accessory input available to intact animals. As previously reported, the main olfactory input remaining after vomeronasal lesions in naive males was poorly effective in activating medial preoptic area, and these animals had impaired mating behavior. The change in access of chemosensory input to medial preoptic area after experience suggests that an experience-dependent synaptic modulation in this pathway, perhaps in the amygdala, may underlie some changes in mating behavior with experience.

Pheromonal male-induced diestrus and cyclicity in aging intact and young estrogenized female rats.

Reproductive aging in female rats is characterized by irregular estrous cycles, repeated pseudopregnancies, persistent estrus, and a decline in fertility. The persistently estrous rats do not ovulate or show cyclic increases of LH despite continued ovarian follicular development. These rats ovulate after mating with fertile males. On the other hand, a single injection of estradiol to young female cyclic rats causes persistent anovulatory estrus similar to that of aging rats, which has suggested that young cyclic estrogen-primed females may be a model for the study of reproductive senescence. We attempted to determine whether the male factor that elicits ovulation in aging females is pheromonal in nature. The effect of such a pheromonal factor on the persistent estrus of young estrogenized females was also tested. We observed the estrous cycle of aging (7-13-mo-old) and young estrogenized female rats daily. Nasal spraying of urine obtained from young adult male rats induced diestrus and regular cyclicity in both aging intact and young estrogenized female rats, probably because of due to pheromones contained in male urine. This fact suggests an important role of chemosensory communication in prolongation of the period with normal estrous cycles in the female's reproductive life.

Sterilization by irradiation of Cadra coutella (Wlk.) (Lepidoptera, Pyralidae) males increased by female sex pheromone.

PIP: Sterilization by irradiation was studied in 0-24 hour old males of Cadra cautella (Ephestia cautella) exposed to 40 Krad of gamma radiation from a 60 cobalt source at a rate of 2250-2200 rad/minute in the presence of female sex pheromone. This radiation dose failed to induce the desired degree of sterility in the absence of pheromone. Pheromone was extraced from the virgin females and applied to filter paper at doses of .1, .5, and 1 female equivalent which was then introduced into containers containing 15-18 males 3 minutes prior to irradiation. Excitation and increased activity of the insects was observed when compared to the controls without the pheromone. The proportion of low percentage hatch increased with increased pheromone presence. The difference between absence and presence of pheromone was marked. It is concluded that these results are evidence of the value of this approach of preconditioning to irradiation when induction of sterility is the objective.^ieng