Effect of Central Injection of Neostigmine on the Bacterial Endotoxin Induced Suppression of GnRH/LH Secretion in Ewes during the Follicular Phase of the Estrous Cycle.

Induced by a bacterial infection, an immune/inflammatory challenge is a potent negative regulator of the reproduction process in females. The reduction of the synthesis of pro-inflammatory cytokine is considered as an effective strategy in the treatment of inflammatory induced neuroendocrine disorders. Therefore, the effect of direct administration of acetylcholinesterase inhibitor-neostigmine-into the third ventricle of the brain on the gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretions under basal and immune stress conditions was evaluated in this study. In the study, 24 adult, 2-years-old Blackhead ewes during the follicular phase of their estrous cycle were used. Immune stress was induced by the intravenous injection of LPS Escherichia coli in a dose of 400 ng/kg. Animals received an intracerebroventricular injection of neostigmine (1 mg/animal) 0.5 h before LPS/saline treatment. It was shown that central administration of neostigmine might prevent the inflammatory-dependent decrease of GnRH/LH secretion in ewes and it had a stimulatory effect on LH release. This central action of neostigmine is connected with its inhibitory action on local pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)α synthesis in the hypothalamus, which indicates the importance of this mediator in the inhibition of GnRH secretion during acute inflammation.

Neuroestradiol in regulation of GnRH release.

Contribution to Special Issue on Fast effects of steroids. The concept that the positive feedback effect of ovarian estradiol (E2) results in GnRH and gonadotropin surges is a well-established principle. However, a series of studies investigating the rapid action of E2 in female rhesus monkeys has led to a new concept that neuroestradiol, synthesized and released in the hypothalamus, also contributes to regulation of the preovulatory GnRH surge. This unexpected finding started from our surprising observation that E2 induces rapid stimulatory action in GnRH neurons in vitro. Subsequently, we confirmed that a similar rapid stimulatory action of E2 occurs in vivo. Unlike subcutaneous injection of E2 benzoate (EB), a brief (10-20 min), direct infusion of EB into the median eminence in ovariectomized (OVX) female monkeys rapidly stimulates release of GnRH and E2 in a pulsatile manner, and the EB-induced GnRH and E2 release is blocked by simultaneous infusion of the aromatase inhibitor, letrozole. This suggests that stimulated release of E2 is of hypothalamic origin. To further determine the role of neuroestradiol we examined the effects of letrozole on EB-induced GnRH and LH surges in OVX females. Results indicate that letrozole treatment greatly attenuated the EB-induced GnRH and LH surges. Collectively, neuroestradiol released from the hypothalamus appears to be necessary for the positive feedback effect of E2 on the GnRH/LH surge.

[The role of inositol deficiency in the etiology of polycystic ovary syndrome disorders]. / Rola niedoboru inozytolu w patofizjologii zaburzen wystepujacych w zespole policystycznych jajników.

Inositol acts as a second messenger in insulin signaling pathway Literature data suggest inositol deficiency in insulin-resistant women with the polycystic ovary syndrome. Supplementation of myo-inisitol decreases insulin resistance as it works as an insulin sensitizing agent. The positive role of myo-inositol in the treatment of polycystic ovary syndrome has been of increased evidence recently The present review presents the effects of myo-inositol on the ovarian, hormonal and metabolic parameters in women with PCOS.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes.

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from -2 to -0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1µmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHß and GnRH-R in the pituitary. In the case of the GnRH and LHß genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

Dehydroepiandrosterone supplementation increases baseline follicular phase progesterone levels.

The use of dehydroepiandrosterone (DHEA) supplementation in infertile patients with diminished ovarian reserve (DOR) has become increasingly popular. It has been our observation that serum progesterone levels during the follicular phase are often increased during controlled ovarian stimulation when DHEA is coadministered. Our aim was to compare progesterone levels during the follicular phase before and during DHEA supplementation in women with DOR undergoing in vitro fertilization (IVF). In a case-control study, we compared progesterone levels during the follicular phase in IVF cycles before and during DHEA supplementation in 15 women with DOR who received 75 mg of DHEA daily. Progesterone levels on stimulation day 5 (0.5 ± 0.29 ng/ml vs. 1.54 ± 0.49 ng/ml; p < 0.0001) and on the day of human chorionic gonadotropin administration (0.75 ± 0.31 ng/ml vs. 1.87 ± 0.49 ng/ml; p < 0.0001) were significantly higher during DHEA treatment. The number of retrieved and fertilized oocytes was similar in both the groups. DHEA administration during IVF cycles in women with DOR causes a significant elevation of progesterone levels without an apparent deleterious effect on cycle outcome.

Menstrual cycle and cue reactivity in women smokers.

INTRODUCTION: Emerging research suggests potential effects of the menstrual cycle on various aspects of smoking behavior in women, but results to date have been mixed. The present study sought to explore the influence of menstrual cycle phase on reactivity to smoking in vivo and stressful imagery cues in a sample of non-treatment-seeking women smokers. METHODS: Via a within-subjects design, nicotine-dependent women (N = 37) participated in a series of four cue reactivity sessions, each during a distinct biologically verified phase of the menstrual cycle (early follicular [EF], mid-follicular [MF], mid-luteal [ML], and late luteal [LL]). Subjective (Questionnaire of Smoking Urges-Brief; QSU-B) and physiological (skin conductance and heart rate) measures of craving and reactivity were collected and compared across phases. RESULTS: Subjective reactive craving (QSU-B) to smoking in vivo cues varied significantly across the menstrual cycle (p = .02) and was higher in both EF and MF phases versus ML and LL phases, but this finding was not sustained when controlling for reactivity to neutral cues. Heart rate reactivity to stressful imagery cues (p = .01) and skin conductance reactivity to smoking in vivo cues (p = .05) varied significantly across the menstrual cycle upon controlling for reactivity to neutral cues, with highest reactivity during the MF phase. DISCUSSION: Menstrual cycle phase may have an effect on reactivity to smoking-related and stressful cues among women smokers. These findings contribute to an expanding literature, suggesting menstrual cycle effects on smoking behaviors in women.

Exercise, sex, menstrual cycle phase, and 17beta-estradiol influence metabolism-related genes in human skeletal muscle.

Higher fat and lower carbohydrate and amino acid oxidation are observed in women compared with men during endurance exercise. We hypothesized that the observed sex difference is due to estrogen and that menstrual cycle phase or supplementation of men with 17beta-estradiol (E(2)) would coordinately influence the mRNA content of genes involved in lipid and/or carbohydrate metabolism in skeletal muscle. Twelve men and twelve women had muscle biopsies taken before and immediately after 90 min of cycling at 65% peak oxygen consumption (Vo(2peak)). Women were studied in the midfollicular (Fol) and midluteal (Lut) phases, and men were studied after 8 days of E(2) or placebo supplementation. Targeted RT-PCR was used to compare mRNA content for genes involved in transcriptional regulation and lipid, carbohydrate, and amino acid metabolism. Sex was the greatest predictor of substrate metabolism gene content. Sex affected the mRNA content of FATm, FABPc, SREBP-1c, mtGPAT, PPARdelta, PPARalpha, CPTI, TFP-alpha, GLUT4, HKII, PFK, and BCOADK (P < 0.05). E(2) administration significantly (P < 0.05) affected the mRNA content of PGC-1alpha, PPARalpha, PPARdelta, TFP-alpha, CPTI, SREBP-1c, mtGPAT, GLUT4, GS-1, and AST. Acute exercise increased the mRNA abundance for PGC-1alpha, HSL, FABPc, CPTI, GLUT4, HKII, and AST (P < 0.05). Menstrual cycle had a small effect on PPARdelta, GP, and glycogenin mRNA content. Overall, women have greater mRNA content for several genes involved in lipid metabolism, which is partially due to an effect of E(2).

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes.

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.

Adding phytoestrogens to clomiphene induction in unexplained infertility patients--a randomized trial.

This study investigated the role of oral phytoestrogens in improving pregnancy rate and cycle outcomes with clomiphene citrate. Patients with unexplained infertility and recurrent clomiphene citrate induction failure, were randomly divided into two groups: group I (n = 60) and group II (n = 59). Both groups received clomiphene citrate 150 mg per day (days 3 to 7). Group I received additional oral phytoestrogen (Cimicifuga racemosa) 120 mg/day from days 1 to 12. Human chorionic gonadotrophin (HCG) injection (10,000 IU i.m.) was given and timed intercourse was recommended when a leading follicle reached >17 mm and serum oestradiol exceeded 200 (pg/ml). There was a non-significant shortening of induction cycles in group I. Oestradiol and LH concentrations were higher in group I compared with group II. Endometrial thickness, serum progesterone and clinical pregnancy rate were significantly higher in group I (8.9 +/- 1.4 mm versus 7.5 +/- 1.3 mm, P < 0.001; 13.3 +/- 3.1 ng/ml versus 9.3 +/- 2.0 ng/ml, P < 0.01; 36.7% versus 13.6%, P < 0.01, respectively). It is concluded that adding C. racemosa rhizome dry extract to clomiphene citrate induction can improve the pregnancy rate and cycle outcomes in these couples.

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers.

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n=20) originating from a common herd were assigned to three treatment groups using a 3 x 3 latin square design replicated in time (3 x) and space (6 x complete and 1x incomplete): (1) heifers received no added mineral to their diet (control; n=53); (2) heifers received a commercially available organic mineral supplement (organic; n=52); or (3) heifers received an all inorganic mineral supplement (inorganic; n=55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1+/-0.7, 3.8+/-0.7, and 3.3+/-0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P<0.05) for inorganic (2.3+/-0.5) and control (1.6+/-0.5) treated heifers than organic (0.4+/-0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.

Simultaneous increases of leptin and gonadotropin-releasing hormone following exogenous estrogen administration in women with normally menstrual cycle.

The aim of this study was to investigate whether administration of exogenous estrogen affects the changes of leptin and GnRH levels in women with normal menstrual cycle. A total of 18 women received a bolus intravenous injection of 20 mg conjugated estrogen (premarin group) at 0800 during the fifth day of menstrual cycle, while another 18 women were administered 20 mL of normal saline as the control group. Fasting blood samples were collected at 0, 4, 8, 24, 28, 32, 48, 56, 72 and 96 hours after injection for analyses of leptin, GnRH, estrone (E(1)), estradiol (E(2)), LH and FSH. Both the mean plasma levels of E(1) and E(2) were significantly increased from 4 hours and significantly sustained elevated levels up to 72 hours after injection of premarin. Simultaneous significant increases of leptin and GnRH levels were observed at 28, 32 and 48 hours after injection, while the controls remained constant. The mean LH and FSH levels were initially suppressed and then significantly increased at 56 and 72 hours after premarin administration. Leptin appears to be involved in the regulation of positive feedback mechanism of estrogen by conveyance of metabolic signal to affect the release of GnRH in hypothalamus, while its participation in the modulation of negative feedback remains unknown.

Effects of isoflavone supplement on healthy women.

Effects of the isoflavone supplement on hormonal states in young premenopausal women were studied by cross-over study design. Administration of 20 mg or 40 mg isoflavones (IF) by tablets, of which 1 g contained 43.5 mg daidzein, 6.0 mg genistein, 24.0 mg glycitein, to 40 young female students for one month caused a prolonged menstruation in 60% of young women, shortened menstruation in 20% of young women, 17% remained unchanged and 3% became irregular. Larger dose tended to elongate more, but 17beta-estradiol levels in both follicular and luteal phages were not different between 20 mg and 40 mg isoflavone intake. Equor excreters tended to show low plasma progesteron level in the luteal phase. Detailed hormonal analysis on 3 of students by a cross over study design showed decreased level of 17beta-estradiol throughout the menstruation cycle. SHBG significantly increased about 10% in all three. DEAS, androstendione, and testosterone showed different responses according to the follicular or luteal phase. T3 and T4 increased as a result of isoflavone tablet administration in the follicular phase, but it decreased in the luteal phase. These changes suggest that isoflavones influence not only estrogen receptor-related functions but the hypothalamo-hypophysis-gonadal axis.

Effect of estrogen on hypothalamic transforming growth factor alpha and gonadotropin-releasing hormone gene expression in the female rhesus monkey.

In order to study whether hypothalamic transforming growth factor alpha (TGFalpha) gene expression in the monkey is estrogen-sensitive, long-term ovariectomized rhesus macaques were implanted subcutaneously with either estradiol-containing (n = 3) or blank (n = 3) Silastic capsules. Blood samples were collected every other day while the animals were lightly sedated with ketamine hydrochloride to monitor circulating LH and estradiol concentrations. Animals were killed with a lethal dose of pentobarbital sodium after a marked suppression of LH secretion was confirmed (81 days of estradiol treatment); the preoptic area (POA), mediobasal hypothalamus (MBH) and samples of cerebral cortex were dissected out, snap-frozen in liquid nitrogen and processed for the determination of TGFalpha messenger RNA (mRNA) by ribonuclease protection assay using a cRNA probe. The opportunity was also taken to study the action of estrogen on hypothalamic GnRH mRNA levels. Although circulating estradiol concentrations of 50-150 pg/ml achieved in the steroid-treated group produced a decrease in hypothalamic GnRH mRNA levels, which was significant in the MBH, TGFalpha mRNA levels in this hypothalamic region and in the POA were not influenced by estrogen treatment. These findings indicate that TGFalpha is probably not involved in mediating the inhibitory action of estradiol on GnRH neurons. Additionally, the relevance of our results to the understanding of the neurobiological mechanisms underlying the initiation of puberty in primates is discussed.

Effects of hachimijiogan, tokishakuyakusan, keishibukuryogan, ninjinto and unkeito on estrogen and progesterone secretion in preovulatory follicles incubated in vitro.

Ovarian follicles, removed from 10-week old rats at 1630 hours diestrus-2, 1100 and 2300 hours proestrus, were incubated for 120 minutes with various doses of Hachimijiogan (HJ), Tokishakuyakusan (TS), Keishibukuryogan (KB), Ninjinto (NT) and Unkeito (UT). The estradiol-17 beta (E2) and progesterone concentrations in the incubation medium were measured. The concentrations of E2 were significantly decreased with TS and KB by growing follicles and with HJ, TS and KB by preovulatory follicles before a LH surge. In contrast, the levels of progesterone were significantly increased with HJ, TS, KB and UT by preovulatory follicles before a LH surge. These results suggest that HJ, TS, KB or UT stimulates preovulatory follicles before a LH surge to secrete progesterone, but TS or KB suppresses E2 secretion by growing preovulatory follicles before a LH surge.

Effects of tokishakuyakusan and keishibukuryogan on steroidogenesis by rat preovulatory follicles in vivo.

The effect of Tokishakuyakusan (TS) and Keishibukuryogan (KB) on estradiol-17 beta, progesterone and testosterone in serum and ovarian tissue from PMS-treated immature rats was examined in vivo. The in vivo intravenous administration of 20 micrograms of TS or KB increased the concentrations of estradiol-17 beta, progesterone and testosterone. These results suggest that TS and KB stimulate in vivo the production of estradiol-17 beta, progesterone and testosterone by preovulatory follicles, and that TS stimulation is more effective than KB.

Dopamine in hypophysial stalk blood of the rhesus monkey and its role in regulating prolactin secretion.

Numerous studies are suggestive of dopamine serving as the hypothalamic PRL-inhibiting factor in the monkey. In the present study, we measured dopamine concentrations in plasma collected from the hypophysial stalk and determined whether those concentrations were sufficient to account for the inhibiting effect on PRL secretion exerted by the hypothalamus. First, we collected hypophysial stalk blood from seven follicular phase monkeys (four anesthetized with pentobarbital and three with phencyclidine) using a transorbital surgical approach. Dopamine concentrations, measured with a liquid chromatographic-electrochemical procedure, averaged 0.76 ng/ml in stalk plasma and less than 0.1 ng/ml in peripheral plasma collected contemporaneously. Next, we determined the rate of dopamine infusion required to produce peripheral plasma concentrations of dopamine similar to those measured in hypophysial stalk plasma. In seven monkeys, a dopamine infusion rate of 0.1 microgram/kg BW . min produced plasma dopamine concentrations of 0.62 ng/ml, whereas a 10-fold higher rate (1.0 microgram/kg . min) produced plasma concentrations of 1.95 ng/ml. Then, we infused these doses of dopamine into intact follicular phase animals, stalk-transected animals, and estrogen-treated stalk-transected animals to determine their effect on PRL release. The physiological dose of dopamine (0.1 microgram/kg . min) significantly suppressed plasma PRL levels in intact follicular phase animals and estrogen-treated stalk-transected animals but not in untreated stalk-transected animals. The higher rate of dopamine infusion (1.0 microgram/kg . min) was required to inhibit PRL release in the latter group. These results demonstrate that dopamine is secreted by the hypothalamus into hypophysial portal blood in quantities sufficient to account for much of the PRL-inhibiting activity known to be caused by the hypothalamus. Moreover, the results suggest that estrogen reinforces the inhibitory effect of dopamine on PRL release in primates, in contrast to its antagonistic effect in rodents. (Endocrinology 108: 489, 1981)

Estrogen-induced gonadotropin surges in female rhesus monkeys after pituitary stalk section.

In order to investigate the primary site of action of estradiol, whether pituitary or hypothalamic, gonadotropin responses to estrogen were studied in female rhesus monkeys before and immediately after pituitary stalk section. The estrogen challenge, consisting of either an injection of estradio benzoate (400 microgram) or an implant of three silastic capsules containing 17 beta-estradiol, was initiated on days 2--5 of the menstrual cycle. The estrogen was given not later than 8 h after stalk section. Estrogens induced LH surges in all five animals before and after stalk section. FSH increases were observed in four of five intact and three of four stalk-sectioned animals. Mean FSH and LH levels in three stalk-sectioned animals treated with oil alone did not differ significantly from preinjection controls. These experiments suggest that the locus of estrogens on gonadotropin release in the rhesus monkey may well reside within the pituitary gland itself.