Sex differences in oxytocin receptor binding in forebrain regions: Correlations with social interest in brain region- and sex- specific ways

Social interest reflects the motivation to approach a conspecific for the assessment of social cues and is measured in rats by the amount of time spent investigating conspecifics. Virgin female rats show lower social interest towards unfamiliar juvenile conspecifics than virgin male rats. We hypothesized that the neuropeptide oxytocin (OT) may modulate sex differences in social interest because of the involvement of OT in pro-social behaviors. We determined whether there are sex differences in OT system parameters in the brain and whether these parameters would correlate with social interest. We also determined whether estrus phase or maternal experience would alter low social interest and whether this would correlate with changes in OT system parameters. Our results show that regardless of estrus phase, females have significantly lower OT receptor (OTR) binding densities than males in the majority of forebrain regions analyzed, including the nucleus accumbens, caudate putamen, lateral septum, bed nucleus of the stria terminalis, medial amygdala, and ventromedial hypothalamus. Interestingly, male social interest correlated positively with OTR binding densities in the medial amygdala, while female social interest correlated negatively with OTR binding densities in the central amygdala. Proestrus/estrus females showed similar social interest to non-estrus females despite increased OTR binding densities in several forebrain areas. Maternal experience had no immediate or long-lasting effects on social interest or OT brain parameters except for higher OTR binding in the medial amygdala in primiparous females. Together, these findings demonstrate that there are robust sex differences in OTR binding densities in multiple forebrain regions of rats and that OTR binding densities correlate with social interest in brain region- and sex-specific ways.     

N,N-Dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines as potent factor Xa inhibitors

A class of N,N-dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines has been discovered as potent factor Xa inhibitors with desirable in vitro and in vivo anticoagulant activity, but with low oral bioavailability. The 5-chloroindole and 6-chlorobenzo[b]thiophene groups are optimal as the factor Xa S1 binding elements. The strategy of incorporating a side chain on the piperazine nucleus to enhance binding affinity has been examined.     

Estrogen effects on the synaptology and neural membranes of the rat hypothalamic arcuate nucleus

The concept of estrogen-induced "sexual differentiation of the brain" has been a useful focus for investigation. However, it has become clear that the action of estrogen on the rat brain is lifelong, including effects on neurogenesis in the fetus, synaptogenesis in the newborn, and synaptic remodeling in the adult. Estrogen imparts sex differences in the rat's brain by shaping synaptology, postsynaptic membranes, and glia within the arcuate nucleus. These effects of estrogen on the arcuate nucleus also could underlie sexual maturation in both sexes and the development of senescent constant estrus in females.     

Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats

Oxytocin (OT) and arginine-8-vasopressin (AVP) were measured by radioimmunoassay in micropunched hypothalamic neurosecretory nuclei of estrous cycling female Sprague-Dawley rats. In the paraventricular nucleus (PVN): the concentration (pg/microgram protein) of OT was significantly higher in rats in diestrus than during proestrus, estrus, or metestrus, while the concentration during metestrus was significantly greater than in proestrus and estrus; the concentration of AVP was significantly lower in animals in estrus than during the other three stages; because the paraventricular OT levels dropped before proestrus, the AVP/OT ratio was significantly greater in animals in proestrus than in diestrus, metestrus, and estrus. In the supraoptic nucleus (SON) a similar trend was noted: the concentration of OT was highest during diestrus, and AVP was lowest during estrus, though neither was significantly different from other stages. Because the OT and AVP cycles in the SON were asynchronous, the ratio of AVP to OT was significantly higher in proestrus than in metestrus or diestrus and significantly greater in estrus than during diestrus. In contrast to these two areas, peptide concentrations did not vary significantly across the estrous cycle in other sites of nonapeptide synthesis, i.e. the anterior commissural nucleus (ACN) and the suprachiasmatic nuclei (SCN).     

5HT7 receptors in the rodent suprachiasmatic nucleus

Serotonergic modulation of circadian rhythms in rodent model preparations has received considerable attention over the past decade. Investigators have also been trying to determine which of the many serotonin receptor subtypes may be mediating the effects of serotonin in the suprachiasmatic nucleus, the location of the biological clock that generates the circadian rhythms. A single study in 1993 using the in vitro rat hypothalamic slice preparation suggested that serotonergic modulation of circadian rhythms at the level of the suprachiasmatic nucleus was acting via the newly discovered 5HT7 receptor subtype. Since that initial claim, serotonin modulation of circadian rhythms at the level of the suprachiasmatic nucleus has generally been attributed to 5HT7 receptor activation. However, when trying to cite relevant literature in support of 5HT7 involvement, it becomes evident that attributing rhythm-related serotonin activity in the suprachiasmatic nucleus to 5HT7 receptors may be somewhat premature. There are issues related to pharmacological specificity, species-specific results, and significant knowledge gaps that necessitate a careful review of the literature to make a judgment as to whether 5HT7 receptors are responsible for serotonergic activity in the rodent suprachiasmatic nucleus. In addition, there is sufficient data available at present to make an initial determination as to the degree of 5HT7 receptor involvement at any level in the generation or modulation of circadian rhythms in rodent species.     

Specific 3H-PGE1 binding sites in rat ovary in estrous cycle and pregnancy.

Stimulation of cAMP synthesis by prostaglandins E series in the rat ovary is consistent with the presence of a prostaglandin receptor in this tissue. Prostaglandin binding sites with specificity for PGE1 in vitro incubation systems have been demonstrated in rat ovary slices and corpora lutea. The binding of 3H-PGE1 was progressively inhibited with increasing amounts of unlabelled PGE1 and PGE2. PGF2alpha inhibitory effect was markedly smaller than that of PGE. 3H-PGE1 binding to the ovary was higher in 3-day-old rats than in 5-day-old and adult animals, when the highest binding was present in estrus. The specific binding of 3H-PGE1 to rat corpora lutea (CL) decreased on days 11 and 13 of pregnancy and then gradually returned to the level found on day 1 during the second half of gestation. This binding of labelled prostaglandin during pregnancy has been studied in relation to the PGE1 stimulation of cAMP synthesis in rat corpora lutea, but no consistent changes were observed in responsiveness.     

Hormone-sensitive lipase expression and IHC localization in the rat ovary, oviduct, and uterus

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.     

Spinal effects of oxytocin on uterine motility in anesthetized rats

The rat uterus receives an innervation from the lumbosacral and thoracolumbar segments of the spinal cord. These segments receive descending oxytocinergic projections from the paraventricular nucleus of the hypothalamus. We tested the hypothesis that oxytocin regulates uterine motility through a spinal site of action. Oxytocin was administered in anesthetized female rats either intrathecally at the lumbosacral or thoracolumbar spinal cord levels or intravenously. Uterine activity was revealed by measuring changes of intrauterine pressure using an indwelling balloon placed in one caudal uterine horn. The uterus displayed a spontaneous activity characterized by intrauterine pressure rises, the frequency, amplitude, and duration of which were dependent on the stage of the estrous cycle. Oxytocin delivered at the lumbosacral level affected the frequency (during proestrus, estrus, and diestrus) and amplitude (during proestrus and estrus) of uterine activity. During estrus, oxytocin delivered at the thoracolumbar level affected the frequency, amplitude, and duration of the intrauterine pressure rises. Intravenous oxytocin not only affected intrauterine pressure rises (namely amplitude during proestrus and estrus and frequency and duration during estrus) but also increased the basal tone during estrus. The effects of lumbosacral oxytocin were partly mimicked by the oxytocin agonist [Thr(4),Gly(7)]-oxytocin blocked by the oxytocin receptor antagonist atosiban and by hexamethonium. Arginine vasopressin delivered at the lumbosacral level had no effect. These results support our hypothesis that oxytocin released by descending paraventriculo-spinal pathways and acting on spinal oxytocin receptors modulates the activity of the uterus. This regulation is cycle dependent.     

Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry--increased sensitivity by chemical derivatisation of the glucuronic acid conjugate

Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.     

KASH 'n Karry: the KASH domain family of cargo-specific cytoskeletal adaptor proteins

A diverse family of proteins has been discovered with a small C-terminal KASH domain in common. KASH domain proteins are localized uniquely to the outer nuclear envelope, enabling their cytoplasmic extensions to tether the nucleus to actin filaments or microtubules. KASH domains are targeted to the outer nuclear envelope by SUN domains of inner nuclear envelope proteins. Several KASH protein genes were discovered as mutant alleles in model organisms with defects in developmentally regulated nuclear positioning. Recently, KASH-less isoforms have been found that connect the cytoskeleton to organelles other than the nucleus. A widened view of these proteins is now emerging, where KASH proteins and their KASH-less counterparts are cargo-specific adaptors that not only link organelles to the cytoskeleton but also regulate developmentally specific organelle movements.     

Binding dynamics of isolated nucleoporin repeat regions to importin-beta

The nuclear pore complex, through the interaction of its proteins with transport receptors, controls the transport of large molecules into and out of the cell's nucleus. There is ample evidence for proteins with FG sequence repeats playing an essential role in this control. Previous studies have elucidated binding spots for FG sequence repeats on the surface of the transport receptor importin-beta by X-ray crystallography and mutational studies. Molecular dynamics simulations have been performed to characterize the interaction of FG sequence repeats with the transport receptor. Observed binding spots have been verified and novel sites discovered, suggesting that importin-beta features many more binding spots than suspected so far. The observed binding spots are in accord with several models of nucleocytoplasmic transport, and the large number of binding spots on importin-beta may be necessary for the pore complex to distinguish between importin-beta and inert proteins, and to allow for its passage through the pore.     

RNA processing: splicing and the cytoplasmic localisation of mRNA

An unexpected link has been discovered between pre-mRNA splicing in the nucleus and mRNA localisation in the cytoplasm. The new findings suggest that recruitment of the Mago Nashi and Y14 proteins upon splicing of oskar mRNA is an essential step in the localisation of the RNA to the posterior pole of the Drosophila oocyte.     

Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: evidence for mediation by norepinephrine

Pituitary levels of oxytocin and vasopressin were maximal on the morning of proestrus, declined during estrus, and were lowest on metestrus in cycling female rats. Norepinephrine levels in the paraventricular nucleus were decreased on proestrus and estrus when compared with metestrus-diestrus. Norepinephrine did not vary in the supraoptic nucleus. Administration of estradiol benzoate to ovariectomized rats elevated oxytocin in the pituitary 54 hr later. This elevation was not affected by a subsequent injection of estrogen or progesterone. Estrogen priming did not affect vasopressin levels in the pituitary, but a second injection of estrogen or of progesterone 48 hr later increased vasopressin in the pituitary when measured 6 hr after the second injection. Vasopressin was decreased 30 hr after a second injection of estrogen. The ovarian hormone treatments that elevated pituitary vasopressin decreased steady state levels of norepinephrine in the paraventricular nucleus and reduced the depletion of norepinephrine after administration of the catecholamine synthesis inhibitor α-methyltyrosine, suggesting a decrease in turnover. Ovarian hormones did not affect norepinephrine in the supraoptic nucleus. The present results suggest a role for posterior pituitary hormones in reproductive processes and a role for noradrenergic mechanisms in the paraventricular nucleus in mediating the effects of ovarian steroids on pituitary vasopressin.     

Morphological evidence for neural interactions between leptin and orexin in the hypothalamus

Both leptin and orexin have been recently discovered as peptides involved in feeding regulation. The morphological evidence of neural interaction between leptin and orexin, one considered to inhibit food intake and the other to stimulate it in the central nervous system (CNS), was studied by use of double immunostaining method. The leptin receptor-like immunoreactive (LR-LI) neurons in the hypothalamic arcuate nucleus and ventromedial nucleus were innervated by orexin-like immunoreactive (OX-LI) neurons. The distribution of LR-LI neurons in the hypothalamus was very similar to that of OX-LI neurons. These results may suggest that leptin and orexin are intimately correlated with each other and that they reciprocally regulate feeding at the hypothalamic level.     

Evaluation of a porcine zona pellucida vaccine for the immunocontraception of domestic kittens (Felis catus)

With a seasonally polyestrus breeding structure, the unwanted domestic cat population has proven difficult to control. Various lethal methods have been used in an attempt to lower this population of cats. Recently, humane attempts to control "pest species," such as the feral cat, have focused on immunocontraception. SpayVacTM is a vaccine that uses antibodies raised against porcine (ZP) antigens to prevent fertilization of the ovum. SpayVac, delivered in a single dose, has been evaluated in fallow deer and several species of seals with >90% reduction in fertility and no adverse reactions. This study evaluated the effectiveness of SpayVac in reducing fertility in domestic kittens. Thirty female kittens were treated with SpayVac containing either Freund's complete adjuvant (FCA) or alum, or with a control vehicle. Kittens were monitored for side effects, estrus cycling at maturity, and fecundity. Anti-porcine ZP antibodies were quantified by ELISA. Immunohistochemical assays measured the species specificity of the antibodies produced and IgG binding in vivo. Despite high anti-porcine ZP antibody titers, neither formulation of SpayVac prevented estrus cycling at maturity or reduced fecundity. Immunohistochemical assays indicated that antibodies produced by cats treated with SpayVac recognized porcine ZP, but not feline ZP.