Co‐expression of the calcitonin receptor gene in the hypothalamic kisspeptin neurons in female rats

Purpose

Hypothalamic kisspeptin neurons are considered to play a critical role in regulating mammalian reproduction and integrating humoral and neuronal inputs that control gonadotropin‐releasing hormone (GnRH)/gonadotropin release. The present study aimed to investigate the upstream regulator candidates for kisspeptin neurons.

Methods

Visualized kisspeptin neurons that were taken from the arcuate nucleus (ARC) of Kiss1‐tdTomato rats were subjected to next‐generation sequencing (NGS) analysis. In situ hybridization (ISH) for the calcitonin receptor gene (Calcr) was performed throughout the whole forebrain of ovariectomized wild‐type female rats that had been implanted with a negative feedback level of estrogen, because the Calcr expression was evident in the ARC kisspeptin neurons from the NGS analysis. Then, a double ISH was performed for the Calcr and kisspeptin gene (Kiss1) in the brain regions, containing either the anteroventral periventricular nucleus (AVPV) or ARC of the female rats.

Results

The NGS analysis revealed that the Calcr was highly expressed in the ARC kisspeptin neurons. It was found that the Calcr was co‐expressed in 12% and 22% of the Kiss1‐expressing cells in the ARC and AVPV, respectively.

Conclusion

The present study suggests that calcitonin receptor signaling could be involved in the regulation of reproductive function through the direct control of the ARC and/or AVPV kisspeptin neurons, and then GnRH/gonadotropin release.     

Role of pituitary adenylate cyclase‐activating polypeptide in modulating hypothalamic‐pituitary system

Background

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a multifunctional peptide that is isolated and identified from the ovine hypothalamus, whose effects and mechanisms have been elucidated in numerous studies. The PACAP and its receptor are widely expressed, not only in the hypothalamus but also in peripheral organs.

Methods

The studies on the role of PACAP in the hypothalamic‐pituitary system, including those by the authors, were summarized.

Results

In the pituitary gonadotrophs, PACAP increases the gonadotrophin α‐, luteinizing hormoneβ‐, and follicle‐stimulating hormone β‐subunit expression and the expression of gonadotropin‐releasing hormone (GnRH) receptor and its own receptor, PAC1R. Moreover, a low‐frequency GnRH pulse increases the expression of PACAP and PAC1R more than a high‐frequency GnRH pulse in the gonadotrophs. The PACAP stimulates prolactin synthesis and secretion and increases PAC1R in the lactotrophs. In the hypothalamus, PACAP increases the expression of the GnRH receptors, although it is unable to increase the expression of GnRH in the GnRH‐producing neurons.

Conclusion

The PACAP not only acts directly in each hormone‐producing cell, it possibly might regulate hormone synthesis via the expression of its own receptors or those of other hormones.     

Pulsatile kisspeptin effectively stimulates gonadotropin-releasing hormone (GnRH)-producing neurons

Hypothalamic kisspeptin is integral to the hypothalamic–pituitary–gonadal axis by stimulating gonadotropin-releasing hormone (GnRH) release. GnRH is released from the hypothalamus in a pulsatile manner and determines the output of the gonadotropins. However, the effect of kisspeptin on GnRH-secreting cells remains unknown. In an experiment using static cultures of GT1-7 cells, kisspeptin did not significantly increase GnRH mRNA expression. However, when kisspeptin was administered to the cells in a pulsatile manner, GnRH mRNA expression was significantly increased. Primary cultures of fetal rat brain containing GnRH-expressing neurons responded to kisspeptin and increased GnRH mRNA expression by 1.65?±?0.27-fold in the static condition. When cells were stimulated with kisspeptin in a pulsatile manner, GnRH mRNA expression was increased by up to 2.40?±?0.21-fold. In perifused GT1-7 cells, pulsatile, but not continuous kisspeptin stimulation, effectively stimulated GnRH mRNA expression. To assess the level of stimulation of GnRH neurons by kisspeptin, the expression of c-fos was examined. In GT1-7 cells, kisspeptin stimulation in the static condition failed to increase c-fos mRNA expression. However, pulsatile kisspeptin stimulation increased c-fos mRNA by 2.31?±?0.47-fold. Similar to the phenomenon observed in GT1-7 cells, pulsatile, but not static, kisspeptin stimulation significantly increased c-fos mRNA expression in the primary cultures of fetal rat brain. These observations suggest that pulsatile kisspeptin more effectively stimulates GnRH-producing cells to increase the production of GnRH.     

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

Introduction

Shallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine.

Methods

We investigated the effects of hypoxia and A_2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo).

Results

The adenosine A_2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O₂ compared to untreated control cells. A_2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O₂ concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A_2B adenosine receptor activation in trophoblast cells. Adenosine receptor A_2B stimulation decreased VEGF expression at 2% and 8% O₂ but led to increased levels at 21% O₂.

Conclusions

These data indicate A_2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A_2B in placental development and possibly in the pathophysiology of preeclampsia.     

Cligosiban,A Novel Brain-Penetrant,Selective Oxytocin Receptor Antagonist,Inhibits Ejaculatory Physiology in Rodents

Introduction

Few treatments are available for men with premature ejaculation (PE); oxytocin (OT) receptor antagonism in the central nervous system (CNS) is a potential new approach.

Allopregnanolone,a GABAA receptor agonist,decreases gonadotropin levels in women. A preliminary study

Animal studies suggest regulatory effects on the hypothalamic-pituitary-gonad axis by allopregnanolone, an endogenous gamma-aminobutyric acid A (GABA_A) receptor agonist. Elevated levels of allopregnanolone in women with hypothalamic amenorrhea have been seen. Isoallopregnanolone is an isomer to allopregnanolone, but without GABA_A receptor effects. The purpose of this study was to investigate effects of allopregnanolone and isoallopregnanolone on gonadotropin levels in healthy women of fertile age. Ten women were given allopregnanolone and five women isoallopregnanolone intravenously in follicular phase. Repeated blood samples were drawn during the test day. Main outcomes were changes in serum levels of follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol, and progesterone. Serum-FSH decreased between 5 and 105?min after the allopregnanolone injection (F(16,144)=2.18, p=0.008). Serum-LH was reduced between 5 and 35?min following the allopregnanolone injection (F(16,144)=2.63, p=0.001). Serum-oestradiol and -progesterone were not significantly changed after allopregnanolone injections. No effect on gonadotropin levels were seen after administration of isoallopregnanolone. Allopregnanolone reduces FSH and LH levels in women and the effect might be mediated via a specific GABA_A receptor activation since isoallopregnanolone lacked this effect. Although the number of women was small, the results suggest a regulatory mechanism on the hypothalamic-pituitary-gonadal axis by allopregnanolon.     

Pathophysiology of Chronic Nitric Oxide Synthase Inhibition-Induced Fetal Growth Restriction in the Rat

Objective. To evaluate the pathophysiology of chronic nitric oxide synthase (NOS) inhibition-induced fetal growth restriction (FGR) in the rat. Methods. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) with or without endothelin (ET-1) receptor A (ET_A) antagonist from day 14 to 21 of gestation. In separate groups, ET_A antagonist and/or L-NAME were discontinued on day 18. On day 21 fetal and placental weights, and maternal and fetal plasma nitrate/nitrite (NO_x) were determined. Results. L-NAME led to FGR, and decreased maternal and fetal NO_x. Maternal NO_x was further decreased when ET_A antagonist was co-administered with L-NAME. ET_A antagonism along with L-NAME did not impact fetal growth. Discontinuation of L‐NAME on day 18 resulted in normal fetal and placental growth at day 21 and an increase of maternal NO_x. Simultaneous cessation of both NOS inhibition and ET_A antagonism on day 18 produced FGR at day 21, whereas continuation of ET_A antagonism after discontinuation of L-NAME resulted in normal fetal growth. Conclusions. NOS inhibition in the pregnant rat leads to decreased maternal and fetal nitric oxide (NO) production and FGR. The effects of NOS inhibition on fetal growth are reversible, and are mediated at least in part by ET-1. With chronic NOS inhibition, ET_A antagonism improves but does not normalize fetal growth, and may allow increased access of L-NAME to the fetal compartment. Continued access of L-NAME to the fetal compartment may limit the effect on fetal growth of any therapeutic intervention in this model of FGR.     

Impact of Sleep Deprivation on the Hypothalamic–Pituitary–Gonadal Axis and Erectile Tissue

Introduction

It is unclear how sleep deprivation (SD) exerts a negative effect on men’s health in terms of hypogonadism.

Non‐esterified fatty acid‐associated ability of follicular fluid to support porcine oocyte maturation and development

Purpose

The effect of supplementing maturation medium with follicular fluid (FF) was examined according to its non‐esterified fatty acid (NEFA) content or with a fatty acid mixture (FA‐Mix) on the developmental competence of oocytes, as well as the mitochondrial quality and quantity in the oocytes and cumulus cells.

Method

Porcine oocytes from a slaughterhouse were used.

Results

The FF or FA‐Mix in maturation medium increased the lipid content in both the oocytes and the cumulus cells, but the adenosine triphosphate content was differentially affected. The FF supplementation increased the mitochondrial DNA copy number, survival of cumulus cells, and rate of oocyte development to the blastocyst stage, whereas the FA‐Mix supplementation did not show these effects. The expression levels of GPC4, PFKP, PRDX3, and TFAM in the cumulus cells increased after FF supplementation, but the expression of GJA1 decreased, compared with the cells that were cultured without FF.

Conclusion

Adding FF and FA‐Mix to the maturation medium increased the lipid content in the oocytes and cumulus cells. The effects of FF on the cumulus cells and oocytes were not observed after FA‐Mix supplementation, indicating that the concentration of the NEFAs in the FF are closely associated with an ability to support oocyte maturation and the metabolism of cumulus cells and oocytes.     

理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素、神经肽Y、八肽胆囊收缩素蛋白与基因的影响

目的观察理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素(LP)、神经肽Y(NPY),八肽胆囊收缩素(CCK-8)等蛋白及基因的调控影响,探讨其治疗厌食的作用机制。方法50只大鼠随机分为对照组,低、中、高剂量理脾复方制剂组,硫酸锌组,每组10只。对照组灌胃给予等体积饮用水;硫酸锌组灌胃给予20 mg/kg硫酸锌口服溶液;低、中、高理脾复方制剂组分别灌胃给予2.4、4.8和9.8 g/kg理脾复方制剂,各组每日灌胃1次,连续7 d,末次灌胃后股动脉采血制备含药血清。体外培养大鼠下丘脑神经元细胞,MAP2免疫荧光鉴定细胞。采用CCK-8法筛选对照组、理脾复方制组(低、中、高剂量组),硫酸锌组在0、2、4、6、8、12、24、36、48 h对下丘脑神经元活性的影响,发现在24 h后时,各含药血清组对下丘脑神经元细胞活性开始增强并具有区别性。ELISA检测下丘脑神经元细胞上清液中LP、NPY、CCK-8的含量;Western Blot法及实时荧光定量PCR法分别检测下丘脑神经元细胞LP、NPY、CCK-8蛋白及基因相对表达量。结果细胞鉴定为下丘脑神经元细胞。与对照组比较,低、中、高剂量理脾复方制剂组中下丘脑神经元细胞上清液中LP、NPY、CCK-8含量降低,差异具有统计学意义(P<0.05);中、高剂量理脾复方制剂组下丘脑神经元细胞中LP、NPY、CCK-8蛋白相对表达量降低,差异具有统计学意义(P<0.05);高剂量理脾复方制剂组下丘脑神经元细胞中LP基因相对表达量降低,差异具有统计学意义(P<0.05);低、中、高剂量理脾复方制剂组下丘脑神经元细胞中NPY、CCK-8基因相对表达量降低,差异具有统计学意义(P<0.05)。结论理脾复方制剂治疗可能通过综合调节下丘脑神经元中LP、NPY、CCK-8蛋白及基因表达,实现改善厌食相关症状,但其影响作用与剂量呈负相关性。     

Expression of the gonadotropin receptors during follicular development

Background

Gonadotropins induce follicular development that leads to ovulation and luteinization. In women, the level of gonadotropins, along with the expression of their receptors, changes dynamically throughout the menstrual cycle. This study aimed to clarify the mechanisms underlying these phenomena.

Methods

The literature was reviewed, including that published by the authors.

Main findings (Results)

Follicle‐stimulating hormone receptor expression in the granulosa cells was induced by androgens that were derived from growth differentiation factor‐9‐stimulated theca cells. In the theca cells, luteinizing hormone receptor (LHR) expression was noted from their appearance. In the granulosa cells, follicle‐stimulating hormone (FSH) stimulation was essential for LHR expression. However, FSH alone was not sufficient to respond to the luteinizing hormone (LH) surge for oocyte maturation, ovulation, and subsequent luteinization. To achieve these stages, various local factors that were derived from the granulosa and theca cells in response to FSH and LH stimulation had to work synergistically in an autocrine/paracrine manner to strongly induce LHR expression. Following the LH surge, the LHR expression decreased markedly; miRNAs were involved in this transient LHR downregulation. Following ovulation, LHR expression drastically increased again toward luteinization.

Conclusion

The expression of gonadotropin receptors is controlled by sophisticated and complicated systems; a breakdown of this system could lead to ovulation disorders.     

Influence of multinerve‐sparing,robot‐assisted radical prostatectomy on the recovery of erection in Japanese patients

Purpose

To evaluate in Japanese patients their sexual function after robot‐assisted radical prostatectomy (RARP) and to investigate the influence of the multinerve‐sparing (NS) grade on their sexual function.

Methods

In total, 225 patients were reviewed with localized prostate cancer who underwent RARP at the authors' institution. They underwent RARP >3 months ago, without pre‐ and posthormone therapy and salvage radiation. Self‐administered International Index of Erectile Function (IIEF) questionnaires were used for assessment preoperatively and 1–48 months postoperatively. In all, 129 patients were evaluated with the preoperative IIEF‐Question 1 and who achieved a score of ≥2 by being divided into five NS groups. The recovery rates of erection (postoperative IIEF‐Question 1 score of ≥2) were calculated by using the Kaplan–Meier analysis.

Results

Seventy‐four percent of all the patients had not attempted sexual intercourse, but 60% had felt sexual desire at 24 months postoperatively. In those patients with a preoperative erection, the recovery rate of erection was 58% at 24 months after the RARP. Across the five NS groups, as the procedure was more nerve‐sparing, the recovery rate of erection became significantly higher. The postoperative effects on erection in the bilateral and unilateral NS groups were significantly superior to those in the other NS groups.

Conclusion

In Japanese patients, erection after a RARP is improved with multiNS grade procedures.     

Human Placental Adenosine Receptor Expression is Elevated in Preeclampsia and Hypoxia Increases Expression of the A2A Receptor

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A₁, A_2A, A_2B and A₃. We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A₁, A_2A, A_2B and A₃ adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A_2A adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.     

Sprague‐Dawley and Fischer Female Rats Differ in Acute Effects of Fluoxetine on Sexual Behavior

IntroductionThe selective serotonin reuptake inhibitor (SSRI), fluoxetine, leads to sexual dysfunction in a substantial proportion of women. In studies with the Fischer inbred rat, the 5‐HT_1A receptor has been implicated in this sexual dysfunction. Whether this association with 5‐HT_1A receptors holds for other rat strains is not known.AimThe effects of acute fluoxetine on sexual behavior in two strains of rats that differ in their response to a 5‐HT_1A receptor agonist were examined. Whether the strain difference is comparable in naturally cycling and hormonally primed, ovariectomized rats was determined.MethodsProestrous rats and ovariectomized rats, hormonally primed with estradiol benzoate and progesterone, were treated with varying doses of fluoxetine. Sexual behavior was examined before and after treatment with the SSRI.Main Outcome MeasuresLordosis to mount ratios, lordosis quality, and proceptive behaviors were quantified. Sprague‐Dawley and Fischer females were compared on each of these measures. The IC₅₀ for inhibition of lordosis behavior was determined.ResultsIn both the intact and the hormonally primed, ovariectomized model, Sprague‐Dawley females were less sensitive to the effects of fluoxetine on sexual behavior. In both groups, fluoxetine showed dose dependency in behavioral inhibition, but a higher dose was required for Sprague‐Dawley than for Fischer females. Naturally cycling, proestrous rats required a higher dose of fluoxetine than hormonally primed ovariectomized rats to produce significant inhibition of sexual behavior. Thus, the strain difference in the response to fluoxetine does not parallel strain differences in the response to a 5‐HT_1A receptor agonist.ConclusionsAcute treatment with fluoxetine inhibits lordosis behavior in both Fischer and Sprague‐Dawley females and the strain difference cannot be explained by reported strain differences in the response to a 5‐HT_1A receptor agonist. Fluoxetine's inhibition of female rat sexual behavior may involve effects of the SSRI in addition to activation of the 5‐HT_1A receptor. Miryala CSJ, Hiegel C, and Uphouse L. Sprague‐Dawley and Fischer female rats differ in acute effects of fluoxetine on sexual behavior. J Sex Med **;**:**–**.     

The effects of chronic testosterone administration on hypothalamic gonadotropin-releasing hormone regulatory factors (Kiss1, NKB,pDyn and RFRP) and their receptors in female rats

The effects of androgens on gonadotropin-releasing hormone (GnRH) secretion in females have not been fully established. To clarify the direct effects of androgens on hypothalamic reproductive factors, we evaluated the effects of chronic testosterone administration on hypothalamic GnRH regulatory factors in ovariectomized (OVX) female rats. Both testosterone and estradiol reduced the serum luteinizing hormone levels of OVX female rats, indicating that, as has been found for estrogen, testosterone suppresses GnRH secretion via negative feedback. Similarly, the administration of testosterone or estradiol suppressed the hypothalamic mRNA levels of kisspeptin and neurokinin B, both of which are positive regulators of GnRH, whereas it did not affect the hypothalamic mRNA levels of the kisspeptin receptor or neurokinin-3 receptor. On the contrary, the administration of testosterone, but not estradiol, suppressed the hypothalamic mRNA expression of prodynorphin, which is a negative regulator of GnRH. The administration of testosterone did not alter the rats’ serum estradiol levels, indicating that testosterone’s effects on hypothalamic factors might be induced by its androgenic activity. These findings suggest that as well as estrogen, androgens have negative feedback effects on GnRH in females and that the underlying mechanisms responsible for these effects are similar, but do not completely correspond, to the mechanisms underlying the effects of estrogen on GnRH.     

Impact of Endothelin A Receptor Antagonist Selectivity in Chronic Nitric Oxide Synthase Inhibition-Induced Fetal Growth Restriction in the Rat

Objective: Endothelin receptor A (ET_A) antagonism improves fetal and placental growth and placental perfusion on days 1 and 4, but not day 7 of a 7-day infusion of a nitric oxide synthase (NOS) inhibitor. Our purpose was to evaluate the significance of the degree of ET_A antagonist selectivity on uteroplacental perfusion and fetal growth on day 7 of chronic NOS inhibition. Methods: Timed-pregnant rats were treated with the NOS inhibitor nitro-L-arginine methyl ester (L-NAME, 2.5 mg/kg/h) with and without one of the following ET_A antagonists or their respective vehicles for 7 days beginning on day 14 of gestation: A-127722 (2,000-fold selective for ET_A over ET_B), FR139317 (8,000-fold ET_A-selective), or ABT-546 (28,000-fold ET_A-selective). Uterine and placental perfusion, as well as fetal and placental weight, was evaluated at the 7^th day of treatment (gestation day 21). Results: L-NAME administration resulted in a significant reduction in uterine and placental perfusion as well as fetal and placental growth. In the setting of NOS inhibition, ET_A antagonism did not improve uterine or placental perfusion or fetal growth after 7 days of infusion irrespective of the degree of selectivity of the antagonist used. Conclusions: ET_A antagonism, irrespective of the degree of receptor selectivity, does not improve fetal growth or uteroplacental perfusion on day 7 of chronic NOS inhibition.