两种抗雌激素药物对雌性斑马鱼脂肪代谢的影响

为探究雌激素对雌鱼体内脂肪代谢的影响,研究分别使用50和250 μg/L的来曲唑（Letrozole、LET）与他莫昔芬（Tamoxifen、TAM）两种抗雌激素药物,构建了雌性斑马鱼（Danio rerio）雌激素缺乏模型和雌激素受体竞争抑制模型,并检测两种药物处理后斑马鱼肝脏、内脏和肌肉的甘油三酯（TG）含量变化以及肝脏内雌激素和脂肪代谢相关基因的变化。结果显示,低浓度LET处理后雌鱼肝脏和内脏TG显著上升（P < 0.05）;高浓度TAM处理后肝脏TG含量显著降低（P < 0.05）,其他各组处理TG均无差异。基因mRNA检测结果表明,两种浓度LET和TAM处理的雌性斑马鱼芳香化酶（CYP19A）表达均显著下调（P < 0.05）,低浓度TAM暴露导致雌激素受体（ERα）表达显著下调（P < 0.05）。此外,两种浓度LET处理均引起了脂肪酸合成酶（FAS）表达显著上调,微粒体的TG转运蛋白（MTP）表达显著下调（P < 0.05）;低浓度TAM引起了MTP表达显著下调（P < 0.05）,而高浓度TAM组则引起了MTP表达显著上调（P < 0.05）。综合各相关指标,研究结果表明雌激素确实在雌性斑马鱼脂肪代谢中发挥作用,然而不同程度和方式的雌激素抑制会导致不同的脂代谢失调表现,这提示鱼体内雌激素紊乱所导致的脂代谢失调与雌激素浓度和作用通路上的受阻位点有关,并受到多重因子参与的内分泌调控网络的调节。     

Implications of leptin in neuroendocrine regulation of male reproduction

Obesity is a major health problem contributing to increased subfertility in males, as well as increased morbidity for diseases related to a decline in testosterone production with aging. Leptin is a hormone produced by adipose tissue whose production increases with the amount of body fat. Several studies have supported a relationship between increased leptin production and regulation of reproductive function. Indeed, leptin acts at all levels of the hypothalamus–pituitary–gonadal (HPG) axis in males. However, most of the obese individuals become insensitive to increased endogenous leptin production and develop a functional leptin resistance. This deregulation of leptin signaling might result in abnormal endocrine and reproductive functions. Altered leptin dynamics may contribute to male infertility in different ways, leading to hypogonadism. These include leptin resistance or leptin insufficiency at the hypothalamus and leptin modulation of testicular physiology. In this review, we address the mechanisms of action of leptin at different levels of the HPG axis. Moreover, the influences of leptin on steroidogenesis and spermatogenesis, as well as seasonal variations of leptin's action on male reproduction are discussed.     

Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio)

Background

The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system.

Methodology/Principal Findings

In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified.

Conclusions/Significance

This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish.     

Characterization of the reproductive effects of the anorexigenic VGF-derived peptide TLQP-21: in vivo and in vitro studies in male rats

VGF (nonacronymic) is a 68-kDa protein encoded by the homonymous gene, which is expressed abundantly at the hypothalamus and has been involved in the control of metabolism and body weight homeostasis. Different active peptide fragments are generated from VGF, including TLQP-21. Circumstantial evidence has suggested that VGF might also participate in the control of reproduction. Yet its mechanisms of action and the eventual role of specific VGF-derived peptides on the hypothalamic-pituitary-gonadal (HPG) axis remain unknown. Herein we report a series of studies on the reproductive effects of TLQP-21 as evaluated in male rats by a combination of in vivo and in vitro analyses. Central administration of TLQP-21 induced acute gonadotropin responses in pubertal and adult male rats, likely via stimulation of GnRH secretion, as documented by static incubations of hypothalamic tissue. In addition, in pubertal (but not adult) males, TLQP-21 stimulated LH secretion directly at the pituitary level. Repeated central administration of TLQP-21 to pubertal males subjected to chronic undernutrition was able to ameliorate the hypogonadotropic state induced by food deprivation. In contrast, chronic administration of TLQP-21 to fed males at puberty resulted in partial desensitization and puberty delay. Finally, in adult (but not pubertal) males, TLQP-21 enhanced hCG-stimulated testosterone secretion by testicular tissue in vitro. In summary, our data are the first to document a complex and multifaceted mode of action of TLQP-21 at different levels of the male HPG axis with predominant stimulatory effects, thus providing a tenable basis for the (direct) reproductive role of this VGF-derived peptide.     

Effect of Cyanotoxins on the Hypothalamic–Pituitary–Gonadal Axis in Male Adult Mouse

Background

Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic–Pituitary–Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis.

Methods

Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. Results: MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice.

Conclusions

MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.     

Lack of functional estrogen receptor beta gene disrupts pubertal male sexual behavior

The estrogen receptor-beta (ERbeta) mediates estrogen action in the female gonads, reproductive tract, and central nervous system. In addition, in rats and mice, gonadotropin-releasing hormone (GnRH-I) neurons coexpress ERbeta. Here we asked if ERbeta plays a role in the onset of puberty and in hypothalamic-pituitary-gonadal (HPG) axis function in male mice. We examined mating behavior, testosterone concentrations, steroid negative feedback on gonadotropins, and GnRH-I function in male ERbeta knockout (ERbetaKO) and wild-type (WT) mice. Peripubertal ERbetaKO males displayed their first ejaculation at a significantly older age than WT littermates. Castrated, adult ERbetaKO mice had significantly higher plasma luteinizing hormone (LH) than WT counterparts. Estradiol (E2) treatment reduced LH and follicle stimulating hormone (FSH) concentrations to an equivalent degree in castrates of both genotypes. In three different measures of the adult GnRH-I system, no genotypic differences were observed. These data show that ERbeta plays an important role in the timing of male sexual behavior at puberty, but does not appear to be involved in adult HPG axis functioning. Furthermore, our data suggest that a primary role of ERbeta may be to regulate ejaculatory behavior.     

鲤鱼发育早期HPG轴和GH/IGF轴相关因子的转录起始分析

采用RT-PCR的方法,以不同发育时期的鲤鱼胚胎和幼鱼为材料,研究了与鱼类生殖相关的HPG轴以及与生长相关的GH/IGF轴中GnRH、GtH以及GH、GHR和IGF重要信号分子的转录起始特征.结果显示,sGnRH、cGnRH、GtH-Ⅰβ卢亚基和GHR于鲤鱼胚胎受精后20h开始转录,IGF-1于受精后23h开始转录,GtH-Ⅱβ亚基于受精后26h开始转录,GtH α亚基于受精后46h开始转录,GH于1dph(孵出后第1天)开始转录.其中,GHR和IGF-1均早于GH开始转录,GtH α亚基和β亚基的转录起始时间不同步.研究结果为揭示鲤鱼生殖与生长间的调控机制积累了重要的科学依据.     

Comparing the effects of atamestane, toremifene and tamoxifen alone and in combination, on bone, serum lipids and uterus in ovariectomized rats

Complete estrogen blockade remains under investigation as a means to optimize anti-estrogen therapy in breast cancer thus both the efficacy and end-organ toxicities are of interest with combinations. We hypothesized that a steroidal aromatase inhibitor (AI) atamestane (ATA) alone, and in combination with the anti-estrogens tamoxifen (TAM) or toremifene (TOR) would have beneficial effects in ovariectomized (OVX) rats on key end-organ functions including bone and lipid metabolism and on the endometrium. Significant positive effects on bone were noted with ATA, TOR, TAM, ATA + TOR, or ATA + TAM. TOR, TAM, ATA + TOR, or ATA + TAM caused significant decreases in serum cholesterol and low-density lipoprotein cholesterol whereas ATA had no effect. Uterine weight and epithelium lining height were not increased by ATA but were by TOR and TAM. No significant differences were found in the key parameters outlined above between OVX rats given TOR and ATA + TOR, or TAM and ATA + TAM. Our data show that ATA in combination with TOR or TAM is equivalent to TOR or TAM alone in terms of end-organ effects within a range of clinically relevant doses. Further studies of combinations of AIs with anti-estrogens on end-organ function are merited.     

Flaxseed alone or in combination with tamoxifen inhibits MCF-7 breast tumor growth in ovariectomized athymic mice with high circulating levels of estrogen

Flaxseed (FS) is rich in mammalian lignan precursors and alpha-linolenic acid, which have been suggested as having anticancer effects. Previous studies have shown that 10% FS inhibits the growth of human estrogen-dependent breast cancer (MCF-7) in athymic mice, and it enhances the inhibitory effect of tamoxifen (TAM). This study determined whether the effect of FS, alone or in combination with TAM, is dose dependent, and it explored the potential mechanism of action. Ovariectomized athymic mice with estradiol (E2) supplementation (1.7 mg/pellet, 60-day release) and established MCF-7 tumors were treated with basal diet control (0FS), 5% FS (5FS), 10% FS (10FS), and TAM (TAM/ 0FS; 5 mg/pellet, 60-day release), alone or in combination (TAM/ 5FS and TAM/10FS) for 8 weeks. Compared with control, 5FS and 10FS significantly inhibited tumor growth by 26% and 38%, respectively. TAM/0FS had an effect similar to the 10FS. TAM/ 5FS and TAM/10FS, respectively, induced significant 48% and 43% reductions in tumor size compared with 0FS, and 18% and 10% reductions compared with TAM/0FS. The relative uterine weight was significantly lower in all TAM groups compared with the control. The reduction of tumor growth resulted from decreased cell proliferation and increased cell apoptosis. TAM/ 5FS caused a significantly higher expression of estrogen receptor-alpha (ERalpha) compared with 5FS and TAM/0FS, whereas TAM/10FS had a higher ERalpha than 10FS and TAM/0FS. Compared with the control, progesterone receptor (PgR) expression was significantly reduced in all treatment groups, but insulin-like growth factor-1 (IGF-1) expression was reduced only by 10FS, TAM/5FS and TAM/10FS. Tumor cell proliferation was significantly positively associated with expression of PgR and IGF-1 and negatively associated with apoptosis and ERalpha. Apoptosis was only associated with ERalpha. In conclusion, FS inhibited MCF-7 tumor growth in a dose-dependent manner and enhanced the inhibitory effect of TAM due to the modulation of ER and growth factor signal transduction pathways.     

Precocious puberty: growth and genetics

The precise neuroendocrine mechanisms underlying activation of hypothalamic-pituitary-gonadal (HPG) axis maturation are elusive. The wide age range of pubertal onset among normal individuals throughout the world may suggest that both genetic and environmental factors modulate the timing of puberty. Early activation of the HPG axis, termed central precocious puberty (CPP), causes psychosocial difficulties and may lead to compromised final height, especially if medical intervention is delayed. Although CPP is considered to be idiopathic in the majority of patients, we have recently reported a 27.5% prevalence of familial cases among 147 patients with idiopathic CPP. Segregation analysis of this cohort suggested an autosomal dominant transmission with incomplete sex-dependent penetrance. Allelic variants of candidate genes that regulate the timing of puberty may cause familial CPP. Detection of these genes will provide a tool for identification of children at risk of developing CPP, enabling early intervention with the aim of preventing its distressing outcomes.     

2013 William Allan Award: My Multifactorial Journey

Gonadotropin-releasing hormone (GnRH) neurons originate outside the CNS in the olfactory placode and migrate into the CNS, where they become integral components of the hypothalamic-pituitary-gonadal (HPG) axis. Disruption of this migration results in Kallmann syndrome (KS), which is characterized by anosmia and pubertal failure due to hypogonadotropic hypogonadism. Using candidate-gene screening, autozygosity mapping, and whole-exome sequencing in a cohort of 30 individuals with KS, we searched for genes newly associated with KS. We identified homozygous loss-of-function mutations in FEZF1 in two independent consanguineous families each with two affected siblings. The FEZF1 product is known to enable axons of olfactory receptor neurons (ORNs) to penetrate the CNS basal lamina in mice. Because a subset of axons in these tracks is the migratory pathway for GnRH neurons, in FEZF1 deficiency, GnRH neurons also fail to enter the brain. These results indicate that FEZF1 is required for establishment of the central component of the HPG axis in humans.     

Mild calorie restriction does not affect testosterone levels and testicular gene expression in mutant mice

The hypothalamic-pituitary-gonadal (HPG) axis and the somatotropic axis are influenced by nutritional factors. Calorie restriction (CR) extends lifespan but suppresses both the HPG and the somatotropic axes. Since most CR studies use a fairly severe (40%-60%) reduction of calorie intake, we hypothesized that a milder CR (20%) might not be deleterious to reproduction in male mice. To test this hypothesis, we evaluated the effects of 20% CR on testicular testosterone content and on testicular expression of genes that are relevant to testicular function and reproductive competence, including insulin-like growth factor-I, cytochrome P450 aromatase (Cyp19a1), androgen receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, cytochrome P450c17 and 3-beta-hydroxysteroid dehydrogenase/isomerase. To relate CR effects to the activity of the somatotropic axis, we have used growth hormone-resistant GHR knockout mice as well as transgenic mice overexpressing GH. Mild CR did not affect testosterone levels in testis homogenates and had little effect on expression of the examined genes in the reproductive organs. Altered activity of the GH/insulin-like growth factor-1 axis had a major impact on the parameters analyzed. The results also suggest that expression of several key genes involved in the control of testicular function is preserved under conditions of mild CR and encourage speculation that mild regimens of CR can produce longevity benefits without impairing reproduction.