Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system.

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Synergistic Action of Virgin Coconut Oil and Clomiphene in Reversing Endocrine Dysregulation in Letrozole-Model of Polycystic Ovarian Syndrome in Rats: Role of Nrf2/HMOX-1 Pathway.

Polycystic ovarian syndrome (PCOS) is an endocrine disorder in women's reproductive age. Currently, the pathophysiology of PCOS is unclear, and the limited treatment options are unsatisfactory. Virgin coconut oil (VCO) is functional food oil associated with pharmacological effects in reproductive disorders. Therefore, we aimed to evaluate whether VCO could enhance clomiphene (CLO) therapy against PCOS in female rats. Rats were randomly divided: (1) Control, (2) PCOS model, (3) PCOS + CLO, (4) PCOS + VCO, and (5) PCOS + CLO + VCO. The PCOS was induced via daily letrozole (1 mg/kg, orally) administration for 21 days. After the PCOS induction, CLO, VCO, and CLO + VCO were administered from days 22 to 36. Serum levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, estrogen, progesterone, and prolactin were estimated. Polymerase chain reaction gene expression for nuclear factor-erythroid-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), catalase (CAT), glutathione reductase (GSR), LH receptor (LHr), androgen receptor (AR), tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and caspase-3 were analyzed. The letrozole-induced PCOS caused considerable increases in GnRH, LH, prolactin, estrogen, and testosterone, whereas FSH decreased significantly compared to the control. The gene expression of Nrf2, HO-1, CAT, and GSR were markedly diminished, while IL-1ß, TNF-α, caspase-3, AR, and LHr prominently increased compared to control. Interestingly, the CLO and VCO separately exerted anti-inflammatory and endocrine balance effects. However, VCO-enhanced CLO effect in LH, prolactin and testosterone, Nrf2, HO-1, CAT, GSR, and AR. VCO may synergize with CLO to depress hyperandrogenism and oxidative inflammation in PCOS.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways.

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH). Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression. Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

GnRH replacement rescues cognition in Down syndrome.

At the present time, no viable treatment exists for cognitive and olfactory deficits in Down syndrome (DS). We show in a DS model (Ts65Dn mice) that these progressive nonreproductive neurological symptoms closely parallel a postpubertal decrease in hypothalamic as well as extrahypothalamic expression of a master molecule that controls reproduction-gonadotropin-releasing hormone (GnRH)-and appear related to an imbalance in a microRNA-gene network known to regulate GnRH neuron maturation together with altered hippocampal synaptic transmission. Epigenetic, cellular, chemogenetic, and pharmacological interventions that restore physiological GnRH levels abolish olfactory and cognitive defects in Ts65Dn mice, whereas pulsatile GnRH therapy improves cognition and brain connectivity in adult DS patients. GnRH thus plays a crucial role in olfaction and cognition, and pulsatile GnRH therapy holds promise to improve cognitive deficits in DS.

Activation of PI3K/Akt prevents hypoxia/reoxygenation-induced GnRH decline via FOXO3a.

Recent studies have suggested that the hypothalamus has an important role in aging by regulating nuclear factor-?B (NF-?B)-directed gonadotropin-releasing hormone (GnRH) decline. Moreover, our previous study has shown that ischemia-reperfusion (IR) injury activates NF-?B to reduce hypothalamic GnRH release, thus suggesting that IR injury may facilitate hypothalamic programming of system aging. In this study, we further examined the role of phosphoinositide 3-kinase (PI3K)/Protein kinase B (Akt) pathway, a critical intracellular signal pathway involved in the repair process after IR, in hypoxia-reoxygenation (HR)-associated GnRH decline in vitro. We used GT1-7 cells and primarily-cultured mouse GnRH neurons as cell models for investigation. Our data revealed that the activation of the PI3K/Akt/Forkhead box protein O3a (FOXO3a) pathway protects GnRH neurons from HR-induced GnRH decline by preventing HR-induced gnrh1 gene inhibition and NF-?B activation. Our results further the understanding of the regulatory mechanisms of HR-associated hypothalamic GnRH decline.

Hyperandrogenism induces proportional changes in the expression of Kiss-1, Tac2, and DynA in hypothalamic KNDy neurons.

BACKGROUND: Kisspeptin released from Kiss-1 neurons in the hypothalamus plays an essential role in the control of the hypothalamic-pituitary-gonadal axis by regulating the release of gonadotropin-releasing hormone (GnRH). In this study, we examined how androgen supplementation affects the characteristics of Kiss-1 neurons. METHODS: We used a Kiss-1-expressing mHypoA-55 cell model that originated from the arcuate nucleus (ARC) of the mouse hypothalamus. These cells are KNDy neurons that co-express neurokinin B (NKB) and dynorphin A (DynA). We stimulated these cells with androgens and examined them. We also examined the ARC region of the hypothalamus in ovary-intact female rats after supplementation with androgens. RESULTS: Stimulation of mHypoA-55 cells with 100 nM testosterone significantly increased Kiss-1 gene expression by 3.20 ± 0.44-fold; testosterone also increased kisspeptin protein expression. The expression of Tac3, the gene encoding NKB, was also increased by 2.69 ± 0.64-fold following stimulation of mHypoA-55 cells with 100 nM testosterone. DynA gene expression in these cells was unchanged by testosterone stimulation, but it was significantly reduced at the protein level. Dihydrotestosterone (DHT) had a similar effect to testosterone in mHypoA-55 cells; kisspeptin and NKB protein expression was significantly increased by DHT, whereas it significantly reduced DynA expression. In ovary-intact female rats, DTH administration significantly increased the gene expression of Kiss-1 and Tac3, but not DynA, in the arcuate nucleus. Exogenous NKB and DynA stimulation failed to modulate Kiss-1 gene expression in mHypoA-55 cells. Unlike androgen stimulation, prolactin stimulation did not modulate kisspeptin, NKB, or DynA protein expression in these cells. CONCLUSIONS: Our observations imply that hyperandrogenemia affects KNDy neurons and changes their neuronal characteristics by increasing kisspeptin and NKB levels and decreasing DynA levels. These changes might cause dysfunction of the hypothalamic-pituitary-gonadal axis.

Progesterone supplementation to improve fertility of selected subgroups of lactating cows during the summer and fall.

One major cause of low fertility of cows in the summer is progesterone deficiency. We found that insertion of a controlled intravaginal drug-releasing (CIDR) device containing progesterone after artificial insemination (AI) increases pregnancy per AI (P/AI) in cows with uterine disease and low body condition score after calving. Here, we treated only these two subgroups, during the summer and autumn. Control (n = 191 AI) and treatment (n = 230 AI) cows were inseminated at estrus and the treated group received a CIDR device on day 5 post-AI, for 14 days. Overall analysis of data during the summer and autumn indicated no significant differences between treatment and control groups. Analysis of the summer data only indicated a significant effect of treatment: P/AI was higher in CIDR-treated vs. control groups (34.2% vs. 19.3%; p < .038). Results indicated a 15% increase in P/AI during the summer for CIDR-treated cows in subgroups that had responded positively to the progesterone treatment.

Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams.

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( O 2 · - ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and O 2 · - levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-PX and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E2 ), O 2 · - and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro.

Hypothalamic-pituitary-adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons.

Hypothalamic involvement and the role of progesterone in the NGF-induced LH surge pathway.

To elucidate the mechanism by which nerve growth factor (NGF) influences the LH secretory pathway in camelids, a series of experiments were done to determine the involvement of the hypothalamus (Experiment 1), the role of GnRH neurons (Experiment 2), and the effect of progesterone (Experiment 3) on the NGF-induced LH surge and ovulation in llamas. In Experiment 1, the declining phase of the NGF-induced LH surge was used to determine if the decline is a result of pituitary depletion or hypothalamic unresponsiveness. Female llamas were treated with NGF and, 7 h later, assigned to three groups and given a second dose of NGF (n = 5), a dose of GnRH (n = 5), or saline (n = 6). The LH response was attenuated after the second dose of NGF vs GnRH. In Experiment 2, Fos expression (marker of neuronal activation) in GnRH neurons was examined in the hypothalamus of llamas after NGF or saline treatment (n = 3 per group). Despite an LH surge in the NGF group but not in the saline group, no differences were detected between groups in Fos/GnRH co-expression. In Experiment 3, llamas in low-, medium-, and high-plasma progesterone groups (n = 4 per group) were treated with NGF. The NGF-induced LH surge did not differ among treatment groups. Results from the present study show that the induction of a preovulatory LH surge by NGF may be controlled by a novel pathway involving GnRH neuro-terminals downstream of the hypothalamus and is independent of progesterone influence.

Brain-derived neurotrophic factor (BDNF) affects the activity of the gonadotrophic axis in sheep.

This study aimed to examine the hypothesis that BDNF modulates the activity of the gonadotrophic axis in sheep. Central infusions of BDNF were administered to sexually mature Polish Merino sheep. The sheep were randomly divided into three groups: the control group received intracerebroventricular (ICV) infusions of the vehicle, the BDNF 10 group received ICV infusions of BDNF at 10 µg/480 µL/day, and the BDNF 60 group was infused with BDNF at 60 µg/480 µL/day. A series of four infusions on three consecutive days was performed. Blood samples were collected on days 0 and 3 of the infusions. Immediately after the experiment, all the sheep were slaughtered, and selected structures of the hypothalamus and pituitaries were collected for Real Time RT-qPCR analysis. The collected plasma samples, as well as parts of pituitaries were stored for radioimmunoassay analysis of LH and FSH. Central treatment with exogenous BDNF stimulated GnRH mRNA expression in the preoptic area, as well as GnRH-R mRNA in the pituitary. Furthermore, substantial changes in the KNDy mRNA expression in the mediobasal hypothalamus were observed after the ICV BDNF administration. Additionally, central BDNF infusion caused a decrease in LH concentration and a simultaneous increase in FSH concentration in peripheral blood. Neither pulse amplitude nor pulse frequency for any gonadotrophin was affected in both groups of sheep that received BDNF infusion. Our results revealed that exogenous BDNF affects GnRH and KNDy gene expression and changes in the LH and FSH pituitary cell secretory activities. These findings suggest that BDNF may participate in the mechanism modulating the activity of the gonadotrophic axis at the central level in female sheep.

A randomized controlled clinical trial on the effect of acupuncture therapy in dairy cows affected by pyometra.

Pyometra (PYO) is a reproductive disease characterized by the accumulation of purulent or mucopurulent material within the uterine lumen, in the presence of an active corpus luteum (CL). As the CL continues secreting progesterone, PYO would develop following endometritis. Due to prohibited use of artificial luteolytic hormones in US certified organic dairies, conventional therapies consisting of administration of prostaglandin F2α, are not applicable. The objective of this study was to evaluate the efficacy of two acupuncture procedures on the treatment of persistent CL in cows with PYO. We hypothesized that acupuncture would reduce CL diameter and serum progesterone (P4) concentrations, leading to regression of PYO. Holstein cows with PYO, at an USDA certified organic dairy farm in Northern Colorado, were enrolled in a randomized controlled clinical trial and assigned to 1 of 3 treatments: (1) control pyometra (CP; no treatment; n = 17); (2) electroacupuncture (EAP; n = 15); and (3) laser acupuncture (LAP; n = 15). Each cow received three 9-min (EAP) or 20-min (LAP) acupuncture sessions in alternate days. All study cows had blood samples collected for determination of serum progesterone concentration at d0, and at d2, d4, d11, d18, and d25 after first treatment. The ovaries were scanned by transrectal ultrasonography at -3d, d0, d2, d4, d11, and d18 to determine the diameter of the CL. The effect of treatment in the outcome variables was evaluated by ANOVA and by repeated measures analyses, accounting for baseline data (CL diameter and progesterone serum concentration). Average ± SE change in CL diameter from d0 to d18 were 0.94 ± 1.0 mm, 0 ± 1.0 mm, and - 0.33 ± 1.0 mm for CP, EAP, and LAP, respectively. The repeated measures analysis indicated no significant differences for CL diameter among groups. None of the study cows had serum progesterone values <1 ng/ml by the end of the monitoring period (d25) and average ± SE change from d0 to d25 were - 4.0 ± 1.97 ng/ml, -0.76 ± 2.1 ng/ml, and 3.24 ± 1.9 ng/ml for CP, EAP, and LAP, respectively. The repeated measures analysis indicated no significant differences for serum progesterone concentrations among groups. On farm cow records reviewed 150 d after treatment indicated that 2 cows in EAP and 1 cow in LAP conceived 38 d, 68 d, and 38 d, after treatment completion. In conclusion, acupuncture was not an effective treatment for persistent CL in cows with PYO, during the monitoring period.

Molecular characterization of different preproGnRHs in Astyanax altiparanae (Characiformes): Effects of GnRH on female reproduction.

Gonadotropin-releasing hormone (GnRH) is a key molecule in the initiation of the hypothalamic-pituitary-gonadal axis. Thus, knowledge about GnRH may contribute to the effectiveness of species reproduction. Using a Neotropical tetra Astyanax altiparanae as a fish model species, the GnRH forms were characterized at the molecular level and the role of injected GnRHs in vivo was evaluated. The full-length complementary DNA (cDNA) sequences of preproGnRH2 (612 bp) and preproGnRH3 (407 bp) of A. altiparanae were obtained, and the GnRH1 form was not detected. The cDNA sequences of preproGnRH2 and preproGnRH3 were found to be conserved, but a change in the amino acid at position 8 of the GnRH3 decapeptide of A. altiparanae was observed. All the injected GnRHs stimulated lhß messenger RNA (mRNA) expression but not fshß mRNA expression, and only GnRH2 was able to increase maturation-inducing steroid (MIS) levels and possibly stimulate oocyte release. Furthermore, only GnRH2 was able to start the entire reproductive hormonal cascade and induce spawning.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

Bisphenol A induces Pomc gene expression through neuroinflammatory and PPARγ nuclear receptor-mediated mechanisms in POMC-expressing hypothalamic neuronal models.

Endocrine disrupting chemicals, such as bisphenol A (BPA), have been linked to obesity. However, the direct effect of BPA on the hypothalamic pro-opiomelanocortin (POMC) neurons, which regulate energy homeostasis, remains unexplored. We define the effect of BPA on functionally characterized, POMC-expressing cell models, mHypoA-POMC/GFP-2 and mHypoE-43/5. Exposure to BPA significantly induced the mRNA levels of Pomc in both primary culture and the cell lines. Neuroinflammatory and steroid receptor mRNA levels were assessed to delineate the potential mechanisms, including inflammatory markers Nfκb, Il6 and Iκba, and steroid receptors Esr1, Esr2, Gpr30, Esrrg, and Pparg. Pre-treatment with anti-inflammatory compounds gonadotropin-releasing hormone, and PS1145, an IκB kinase inhibitor, abrogated the BPA-mediated Pomc induction. Furthermore, T0070907, a PPARγ antagonist, abolished Pomc induction, while the GPR30 antagonist G15 had no effect. These findings indicate that BPA may have direct effects on POMC neurons in the hypothalamus, utilizing neuroinflammatory mechanisms and through PPARγ nuclear receptors.

Evaluation of ovarian and metabolic effects of GnRH modulators in two rat models of polycystic ovary syndrome.

Gonadotropin-releasing hormone (GnRH) modulators are widely used in numerous reproductive conditions including infertility. Several clinical studies showed mixed results regarding the efficacy of GnRH modulators in patients with polycystic ovary syndrome (PCOS). Along with this, few preclinical studies focus on the effect of GnRH modulators in PCOS-induced animals. Therefore, the present study was designed to study the effect of leuprolide and cetrorelix on hormonal, metabolic, and menstrual dysfunction PCOS rats. Prepubertal female rats were divided into four groups: Group I received a normal pellet diet and Groups II, III, and IV received 40% high-fat diet for 105 days. Similarly, adult female rats were divided into four groups: Group I received 1% carboxymethylcellulose (CMC) and Groups II, III, and IV received letrozole (1 mg/kg, per oral [p.o.] in 1% CMC) for 21 days. Thereafter, leuprolide (2.5 µg/rat, s.c.) and cetrorelix (10 µg/kg, subcutaneous [s.c.]) treatment were given to Group III and Group IV animals, respectively, for 21 days. Oral glucose tolerance test, lipid profile, fasting glucose, insulin, estrus cycle, hormonal profile, ovary weight, ovarian histopathological changes, and LHR and FSHR expressions were measured. Treatment with leuprolide and cetrorelix did not improve glucose intolerance, insulin level, insulin sensitivity indices, sex hormone levels, lipid profile, and estrus cycle. Only testosterone level, total cholesterol level, and follicular development were improved. Therefore, it was concluded that both leuprolide and cetrorelix showed improvement in follicular development, which could be helpful for improving fertility in PCOS.

The relationship between gonadotropin releasing hormone and ovulation inducing factor/nerve growth factor receptors in the hypothalamus of the llama.

BACKGROUND: A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus. METHODS: Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH. RESULTS: Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons. CONCLUSIONS: Results do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

Up-regulation of DUSP5 and DUSP6 by gonadotropin-releasing hormone in cultured hypothalamic neurons, GT1-7 cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates anterior pituitary gonadotrophs to synthesize and secrete gonadotropins. In addition to gonadotrophs, GnRH neurons also express GnRH receptors, and the autocrine action of GnRH is reportedly involved in the regulation of functions of GnRH neurons. There is accumulating evidence that extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases (MAPKs), is activated by GnRH and involved in various effects of GnRH in GnRH neurons. In the present study, we performed microarray analysis to examine the types of genes whose expression was regulated by GnRH in immortalized mouse GnRH neurons (GT1-7 cells). We found that 257 genes among 55,681 genes examined were up-regulated after 30-min treatment of GT1-7 cells with GnRH. These up-regulated genes included four dual-specificity MAPK phosphatases (DUSPs), DUSP1, DUSP2, DUSP5, and DUSP6. Reverse transcription-polymerase chain reaction analysis confirmed that the mRNA levels of DUSP5 and DUSP6 were robustly increased within 30 min. U0126, an inhibitor of ERK activation, completely inhibited the increases in the mRNA levels of DUSP5 and DUSP6. Immunoblotting analysis revealed that ERK activation peaked at 5 min and declined steeply at 60 min, whereas DUSP5 and DUSP6 proteins were increased from 60 min. It was notable that down-regulation of DUSP6 augmented GnRH-induced ERK activation approximately 1.7-fold at 60 min. These results suggested that the up-regulation of DUSP6 regulates the duration of ERK activation at least in part.

Hypothalamic Response to Kisspeptin-54 and Pituitary Response to Gonadotropin-Releasing Hormone Are Preserved in Healthy Older Men.

BACKGROUND: Male testosterone levels decline by 1% per year from the age of 40 years. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin-54 and the pituitary response to gonadotropin-releasing hormone (GnRH) of older men with those of young men. METHODS: Following 1 h of baseline sampling, healthy older men (n = 5, mean age 59.3 ± 2.9 years) received a 3-h intravenous infusion of either vehicle, kisspeptin-54 0.1, 0.3, or 1.0 nmol/kg/h or GnRH 0.1 nmol/kg/h, on five different study days. Serum gonadotropins and total testosterone were measured every 10 min and compared to those of young men (n = 5/group) (mean age 28.9 ± 2.0 years) with a similar body mass index (24 kg/m2) who underwent the same protocol. RESULTS: Kisspeptin-54 and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (p < 0.001 for all groups). Gonadotropin response to kisspeptin-54 was at least preserved in older men when compared to young men. At the highest dose of kisspeptin-54 (1.0 nmol/kg/h), a significantly greater luteinising hormone (LH) (p = 0.003) response was observed in older men. The follicle-stimulating hormone (FSH) response to GnRH was increased in older men (p = 0.002), but the LH response was similar (p = 0.38). Serum testosterone rises following all doses of kisspeptin-54 (p ≤ 0.009) were reduced in older men. CONCLUSIONS: Our data suggest that healthy older men without late-onset hypo-gonadism (LOH) have preserved hypothalamic response to kisspeptin-54 and pituitary response to GnRH, but impaired testicular response. Further work is required to investigate the use of kisspeptin-54 to identify hypothalamic deficits in men with LOH.

Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHß and LHß in anterior pituitary cells in vitro.

Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.