Stuart Milligan

The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility.

Ovulatory dysfunction is the commonest cause of female infertility. Here we show that the co-repressor nuclear-receptor-interacting protein 1 (Nrip1; encoded by the gene Nrip1) is essential for ovulation. Mice null for this protein are viable, but female mice are infertile because of complete failure of mature follicles to release the oocyte at ovulation. In contrast, luteinization proceeds normally, resulting in a phenotype closely resembling that of luteinized unruptured follicle syndrome, often associated with infertility in women. Therefore, whereas the pre-ovulatory surge of luteinizing hormone induces both ovulation and luteinization, the ability to suppress the action of nuclear receptors is essential for the coordinated control of ovarian function with the essential process of oocyte release dependent on the activity of the transcriptional co-repressor Nrip1 (RIP40).

Kisspeptin signalling in the hypothalamic arcuate nucleus regulates GnRH pulse generator frequency in the rat

Background Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses. Methodology/Principal Findings We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion. Conclusions/Significance These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.

Down‐Regulation of Hypothalamic Kisspeptin and its Receptor, Kiss1r, mRNA Expression is Associated with Stress‐Induced Suppression of Luteinising Hormone Secretion in the Female Rat

Identification of kisspeptin (Kiss1) and its G protein‐coupled receptor 54 (Kiss1r) as an essential component of the hypothalamic‐pituitary‐gonadal (HPG) axis controlling gonadotrophin secretion raises the possibility that kisspeptin‐Kiss1r signalling may play a critical role in the transduction of stress‐induced suppression of reproduction. We examined the effects of: (i) three different stressors, known to suppress pulsatile luteinising hormone (LH) secretion; (ii) corticotrophin‐releasing factor (CRF); and (iii) corticosterone on Kiss1 and Kiss1r expression in key hypothalamic sites regulating gonadotrophin secretion: the medial preoptic area (mPOA) and arcuate nucleus (ARC). Ovariectomised oestrogen‐replaced rats were implanted with i.v., subcutaneous or i.c.v. cannulae. Blood samples were collected at 5‐min intervals for 5–6 h for detection of LH. Quantitative reverse transcriptase‐polymerase chain reaction was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the mPOA and ARC collected 6 h after restraint, insulin‐induced hypoglycaemia or lipopolysaccharide stress, or after i.c.v. administration of CRF, or acute or chronic subcutaneous administration of corticosterone. We observed down‐regulation of at least one component of the kisspeptin‐Kiss1r signalling system by each of the stress paradigms within the mPOA and ARC. CRF decreased Kiss1 and Kiss1r expression in both the mPOA and ARC. Both acute and chronic stress levels of corticosterone resulted in a concomitant decrease in Kiss1 and an increase in kiss1r mRNA expression in the mPOA and ARC. This differential regulation of Kiss1 and Kiss1r might account for the lack of effect corticosterone has on pulsatile LH secretion. Considering the pivotal role for kisspeptin‐Kiss1r signalling in the control of the HPG axis, these results suggest that the reduced Kiss1‐Kiss1r expression may be a contributing factor in stress‐related suppression of LH secretion.

In-vitro activity of S. lavandulaefolia (Spanish sage) relevant to treatment of Alzheimer's disease

Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and individual monoterpenoid constituents have been shown to inhibit the enzyme acetylcholinesterase in‐vitro and in‐vivo. This activity is relevant to the treatment of Alzheimers disease, since anticholinesterase drugs are currently the only drugs available to treat Alzheimers disease. Other activities relevant to Alzheimers disease include antioxidant, anti‐inflammatory and estrogenic effects. Results of in‐vitro tests for these activities are reported here for S. lavandulaefolia extracts, the essential oil and its major constituents. Antioxidant activity (inhibition of bovine brain liposome peroxidation) was found in the EtOH extract of the dried herb (5 mg mL−1) and the monoterpenoids (0.1 M) α‐ and β‐pinene and 1,8‐cineole. Thujone and geraniol had lower antioxidant effects, while camphor had no antioxidant effects. Possible anti‐inflammatory activity (eicosanoid inhibition in rat leucocytes) was found in the EtOH extract (50 μg mL−1) and was shown by the monoterpenoids α‐pinene and geraniol (0.2 mM), but not 1,8‐cineole, thujone or camphor. Possible estrogenic activity (via induction of β‐galactosidase activity in yeast cells) was found in the essential oil (0.01 mg mL−1) and the monoterpenoid geraniol (0.1–2 mM). 1,8‐Cineole, α‐ and β‐pinene and thujone did not exhibit estrogenic activity in this analysis. These results demonstrate that S. lavandulaefolia, its essential oil and some chemical constituents have properties relevant to the treatment of Alzheimers disease and provide further data supporting the value of carrying out clinical studies in patients with Alzheimers disease using this plant species.

The Inhibitory Effects of Neurokinin B on GnRH Pulse Generator Frequency in the Female Rat

Neurokinin B (NKB) and its receptor (neurokinin-3 receptor) are coexpressed with kisspeptin and dynorphin A (Dyn) within neurons of the hypothalamic arcuate nucleus, the suggested site of the GnRH pulse generator. It is thought that these neuropeptides interact to regulate gonadotropin secretion. Using the ovariectomized (OVX) and OVX 17β-estradiol-replaced rat models, we have carried out a series of in vivo neuropharmacological and electrophysiological experiments to elucidate the hierarchy between the kisspeptin, NKB, and Dyn signaling systems. Rats were implanted with intracerebroventricular cannulae and cardiac catheters for frequent (every 5 min) automated serial blood sampling. Freely moving rats were bled for 6 h, with intracerebroventricular injections taking place after a 2-h control bleeding period. A further group of OVX rats was implanted with intra-arcuate electrodes for the recording of multiunit activity volleys, which coincide invariably with LH pulses. Intracerebroventricular administration of the selective neurokinin-3 receptor agonist, senktide (100-600 pmol), caused a dose-dependent suppression of LH pulses and multiunit activity volleys. The effects of senktide did not differ between OVX and 17β-estradiol-replaced OVX animals. Pretreatment with a selective Dyn receptor (κ opioid receptor) antagonist, norbinaltorphimine (6.8 nmol), blocked the senktide-induced inhibition of pulsatile LH secretion. Intracerebroventricular injection of senktide did not affect the rise in LH concentrations after administration of kisspeptin (1 nmol), and neither did kisspeptin preclude the senktide-induced suppression of LH pulses. These data show that NKB suppresses the frequency of the GnRH pulse generator in a Dyn/κ opioid receptor-dependent fashion.

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na+ and CI− ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na+ and CI− channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.

Assessment of estrogenic activity in some common essential oil constituents

Estrogenic responses have not only been associated with endocrine function, but also with cognitive function. Several studies have indicated that estrogen replacement therapy has favourable effects on cognition, and may have potential in the prevention and treatment of Alzheimers disease. Thus, ligands for the estrogen receptor, that have a better efficacy and adverse‐effect profile than drugs currently available, require investigation. This study was undertaken to investigate the potential estrogenic activity of a number of essential oil constituents. Initially, estrogenic activity was determined by a sensitive and specific bioassay using recombinant yeast cells expressing the human estrogen receptor. At high concentrations, estrogenic activity was detected for citral (geranial and neral), geraniol, nerol and trans‐anethole, while eugenol showed anti‐estrogenic activity. Molecular graphics studies were undertaken to identify the possible mechanisms for the interaction of geranial, neral, geraniol, nerol and eugenol with the ligand‐binding domain of the estrogen α‐receptor, using the computer program HyperChem. Citral, geraniol, nerol and eugenol were also able to displace [3H]17β‐estradiol from isolated α‐ and β‐human estrogen receptors, but none of these compounds showed estrogenic or anti‐estrogenic activity in the estrogen‐responsive human cell line Ishikawa Var I at levels below their cytotoxic concentrations, and none showed activity in a yeast screen for androgenic and anti‐androgenic activity. The potential in‐vivo estrogenic effects of citral and geraniol were examined in ovariectomized mice, but neither compound showed any ability to stimulate the characteristic estrogenic responses of uterine hypertrophy or acute increase in uterine vascular permeability. These results show that very high concentrations of some commonly used essential oil constituents appear to have the potential to interact with estrogen receptors, although the biological significance of this is uncertain.

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.

Neonatal Lipopolysaccharide Exposure Delays Puberty and Alters Hypothalamic Kiss1 and Kiss1r mRNA Expression in the Female Rat

Immunological challenge experienced in early life can have long‐term programming effects on the hypothalamic‐pituitary‐adrenal axis that permanently influence the stress response. Similarly, neonatal exposure to immunological stress enhances stress‐induced suppression of the hypothalamic‐pituitary gonadal (HPG) axis in adulthood, but may also affect earlier development, including the timing of puberty. To investigate the timing of the critical window for this programming of the HPG axis, neonatal female rats were injected with lipopolysaccharide (LPS; 50 μg/kg i.p.) or saline on postnatal days 3 + 5, 7 + 9, or 14 + 16 and monitored for vaginal opening and first vaginal oestrus as markers of puberty. We also investigated the effects of neonatal programming on the development of the expression patterns of kisspeptin (Kiss1) and its receptor (Kiss1r) in hypothalamic sites known to contain kisspeptin‐expressing neuronal populations critical to reproductive function: the medial preoptic area (mPOA) and the arcuate nucleus in neonatally‐stressed animals. We determined that the critical period for a significant delay in puberty as a result of neonatal LPS exposure is before 7 days of age in the female rat, and demonstrated that Kiss1, but not Kiss1r mRNA, expression in the mPOA is down‐regulated in pre‐pubertal females. These data suggest that the mPOA population of kisspeptin neurones play a pivotal role in controlling the onset of puberty, and that their function can be affected by neonatal stress.

Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose-binding site of the human erythrocyte glucose transporter GLUT1

This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea. Testosterone, 4‐androstene‐3,17‐dione, dehydroepiandrosterone (DHEA) and DHEA‐3‐acetate inhibit glucose exit from human erythrocytes with half‐maximal inhibitions (Ki) of 39.2±8.9, 29.6±3.7, 48.1±10.2 and 4.8±0.98 μM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR). Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3‐gallate (ECG) has a Ki ECG of 0.14±0.01 μM, and epigallocatechin 3‐gallate (EGCG) has a Ki EGCG of 0.97±0.13 μM. Flutamide reverses these effects. Androgen‐screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action. There are sequence homologies between GLUT1 and the ligand‐binding domain (LBD) of hAR containing the amino‐acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3‐keto and 17‐β OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.