Human-like modes of communication, including mutual gaze, in dogs may have been acquired during domestication with humans. We show that gazing behavior from dogs, but not wolves, increased urinary oxytocin concentrations in owners, which consequently facilitated owners' affiliation and increased oxytocin concentration in dogs. Further, nasally administered oxytocin increased gazing behavior in dogs, which in turn increased urinary oxytocin concentrations in owners. These findings support the existence of an interspecies oxytocin-mediated positive loop facilitated and modulated by gazing, which may have supported the coevolution of human-dog bonding by engaging common modes of communicating social attachment.

GPR54 is a novel G protein-coupled receptor speculated to be essential for sexual development. However, its role in the regulation of GnRH types is unknown. To address this issue, we cloned GPR54 from the brain of a cichlid fish (tilapia Oreochromis niloticus) and determined its expression in immature and mature males using our newly developed technique: laser-captured microdissection of single digoxigenin-labeled GnRH neurons coupled with real-time quantitative PCR. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. Absolute copies of GnRH1 and GnRH3, not GnRH2, mRNAs were significantly high in mature compared with immature males. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P < 0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1, GnRH2, and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain; on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P < 0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence of the structure of a nonmammalian GPR54, which is highly conserved during evolution and is expressed in GnRH1, GnRH2, and GnRH3 neurons. Furthermore, we propose that the expression of GPR54 is a "stop signal" for GnRH1, GnRH2, and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.

Mutual gaze is the most fundamental manifestation of social bonding in humans between mothers and infants and between sexual partners in monogamous species. Dog-to-owner gaze probably evolved as a form of social communication during domestication with humans, leading to the establishment of a human-dog bond that is similar to a mother-infant relationship. Urinary oxytocin increases in mothers following mutual gaze in both mothers and infants. A rise in urinary oxytocin occurs in dogs following mutual gaze, but it is unclear whether the increase also occurs in dog owners.This study investigated the effect of mutual gaze in both dogs and their owners on levels of urinary oxytocin. A primary aim was to determine whether there is a causal relationship between mutual gaze and the release of oxytocin. The authors tested the hypothesis that an oxytocin-mediated positive loop (as has been postulated between mother and infants) exists between humans and dogs that is mediated by gaze. To show that the hormone was a cause not just an effect of the interaction, oxytocin was administered intranasally to dogs, and the gazing interaction between dogs and their owners as well as unfamiliar humans was assessed.Gazing behavior increased urinary oxytocin in dogs as well as their owners. Owners and dogs sharing a long mutual gaze had higher levels of oxytocin in their urine than did owners and dogs with shorter eye contact. Although a prolonged gaze increased oxytocin in dogs, it did not increase levels of oxytoxin in hand-raised pet wolves, suggesting that mutual gaze is not used in wolves as a form of social communication with humans. Female dogs receiving intranasal oxytocin gazed longer at their owners than did those given saline. Moreover, oxytocin levels were increased by intranasal oxytocin in dog owners (who had not been given this hormone). These mutual effects were not seen between dogs and unfamiliar humans or between male dogs and their owners.These findings indicate that oxytocin is the cause, not the effect, of the interaction and support of the existence of an interspecies self-perpetuating oxytocin-mediated positive loop facilitated and modulated by mutual gazing. Gazing behavior may have supported the coevolution of human-dog bonding as a common mode of communicating social attachment.

Striking sex difference was detected in the expression of estrogen receptor (ER)␤E strogen plays critical roles in sexual differentiation of the developing brain and sex-specific regulation of reproductive neuroendocrinology in adults (1, 2). Cellular estrogen signaling is conveyed by nuclear estrogen receptors (ERs), which include the classical ER␣ as well as the recently cloned ER␤ (3). Both ERs are expressed in the preoptic area (POA), hypothalamus and limbic structures, which have been implicated in the regulation of reproduction (4-6). It is unclear, however, whether ER␤, like ER␣ (7), is expressed in a sex-specific manner (8, 9). Furthermore, the presence of both ERs in the same neurons could alter the specificity of the transcription by forming heterodimers (10) and might produce different responses to estrogen in different cells, depending on the ratios of ER␣ and ER␤ (11).Disruption of either ER␣ or ER␤ affects various aspects of reproduction. Female and male ER␣ knockout mice are infertile (12), and ER␤ knockout females have a reduced fecundity (13). Anovulation and polycystic or hemorrhagic ovary are present in the ER␣ knockouts (14). Reductions of ovulatory capacity and polycystic ovary occur in the ER␤ knockouts (13). The syndrome may be caused, at least partially, by the impairment in the central mechanism for the secretion of luteinizing hormone.The anteroventral periventricular nucleus (AVPV) is sexually dimorphic with over three times as many dopaminergic neurons in the female rat compared with males (15), and this supports the importance of this brain region in the control of estrous cyclicity, which is absent in males. Indeed, small lesions confined to the AVPV block the cyclic release of luteinizing hormone in female rat and results in anovulatory, persistent estrous state (16).Implantation of microcannulae containing antiestrogens into the AVPV suppressed spontaneous luteinizing hormone surges (17).In the present study, striking sex difference, detected in the ER␤ expression in the AVPV, was reversed by altering neonatal steroid environment. ER␤ mRNA and ER␣ immunoreactivity colocalized in many AVPV cells, some of which would be dopaminergic in nature. Infusion of ER␤ antisense oligonucleotides prolonged vaginal estrus and was accompanied by a 50% reduction of ER␤ immunoreactivity. Materials and MethodsSubjects. Female and male Sprague-Dawley rats (Saitama Experimental Animals, Saitama, Japan) were used on postnatal d 7, 14, 21, 35, or 60 (d 1 ϭ the day of birth) for brain morphometry. They were maintained in a controlled environment at 23°C with a 12-hr light͞12-h dark cycle (lights on at 11 a.m.). The weaning occurred on d 21. Free access to laboratory chow and water was allowed thereafter. A cohort of animals underwent endocrine manipulations as neonates or pups: males were orchidectomized under hypothermia on d 1; females received daily s.c. injections of 10 g 17␤-estradiol benzoate (Sigma) in oil on d 1 through d 10. All juvenile males and females were used without gonadectomy.ER␤ mRNA ...

Functional analysis of GnRH neurons is limited, although these neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiological analysis of GnRH neurons. To identify GnRH neurons, we tagged the neurons with green fluorescence protein by a transgenic technique. A dispersed culture of GnRH neurons was prepared from the transgenic rats. After overnight culture, a perforated patch clamp was applied to the identified GnRH neurons to analyze the Ca2+ currents. In neonatal GnRH neurons, high voltage-activated Ca2+ currents were clearly observed, but low voltage-activated Ca2+ current was negligible. Nimodipine (L-type channel blocker) and omega-conotoxin GVIA (N-type channel blocker) each attenuated the current by approximately 20%. The R-type channel blocker SNX-482 attenuated the current by approximately 55%. Inhibition by the P/Q-type channel blocker omega-agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca2+ currents were observed. Inhibitions by nifedipine, omega-conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by omega-agatoxin IVA was clearly seen in 40-61% of the GnRH neurons examined. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R-, and T-type channels. Expressions of P/Q- and T-type channels are developmentally regulated.

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.

No abstract

The cognate receptor for any of the known gonadotropin-releasing hormones (GnRHs) has not been directly demonstrated. In order to establish this and shed light on the functions of GnRH types, we analyzed the neuroanatomical location and time of initial expression of three distinct GnRH receptors (GnRH-Rs) and the three endogenous GnRHs in the brain of developing and sexually mature tilapia Oreochromis niloticus using immunocytochemistry. In all age groups, including males and females, GnRH-RIA was seen specifically in gonadotropes (Parhar et al. [2002] J Neuroendocrinol 14:657-665) but was undetectable in the brain. On day 8 after fertilization, GnRH-RIB was first seen in the periventricular hypothalamus (lateral recess nucleus, posterior recess nucleus, posterior tuberal nucleus) and GnRH-RIII in the olfactory epithelium, olfactory bulb, telencephalon, preoptic region, mediobasal hypothalamus, thalamus, mesencephalon, and in the hindbrain. Double-label immunocytochemistry showed GnRH1 (Ser(8) GnRH)-immunoreactive neuronal processes projecting mainly to the proximal pars distalis of the pituitary, while GnRH2 (His(5), Trp(7), Tyr(8) GnRH) and GnRH3 (Trp(7), Leu(8) GnRH) fibers were observed in close association with cells containing GnRH-RIB and GnRH-RIII in the brain. These results suggest that GnRH-RIA might be hypophysiotropic in nature, whereas GnRH-RIB and GnRH-RIII could have additional neuromodulatory functions. Further, evidence of close proximity of GnRH-R-containing cells and neuronal processes of multiple GnRH types suggests complex cross-talk between several GnRH ligands and GnRH-Rs.