Kisspeptin调节黄体生成素分泌的关键部位在弓状核

doi:10.3969/j.issn.1673-5374.2013.02.009

摘要/Abstract

摘要：

Kisspeptin是激活下丘脑-垂体-性腺轴区的重要因子。实验建立促性腺激素释放激素/加强绿色荧光蛋白转基因大鼠。分别于其侧脑室注射Kisspeptin 拮抗剂234肽。弓上核和视前叶区注射1，10，100 pM 小肽Kisspeptin-10。免疫组织化学染色显示，大鼠侧脑室内注射Kisspeptin拮抗剂 234肽后，黄体生成素脉冲式分泌可以被抑制；在大鼠弓状核和内侧视前区内注射小肽Kisspeptin-10后，其黄体生成素脉冲式分泌水平明显增加。ELISA检测显示，在转基因大鼠弓状核内注射100 pM Kisspeptin-10作用1 h后，反映促性腺激素释放激素水平的黄体生成素累积水平明显高于弓状核内10 pM Kisspeptin-10给药组。说明Kisspeptin可直接促性腺激素释放激素/加强绿色荧光蛋白转基因大鼠弓状核和内侧视前区促性腺激素释放激素的分泌和黄体生成素的释放。弓状核是Kisspeptin调节黄体生成素脉冲式分泌的关键部位。

关键词: 神经再生, 生物因子, 促性腺激素释放激素, 加强绿色荧光蛋白, 大鼠, 转基因, 黄体生成素, G蛋白偶联受体54, 内侧视前区, 弓状核, 前腹侧室旁核, 下丘脑终板血管区, 图片文章

Abstract:

Kisspeptin is essential for activation of the hypothalamo-pituitary-gonadal axis. In this study, we established gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. Rats were injected with 1, 10, or 100 pM kisspeptin-10, a peptide derived from full-length kisspeptin, into the arcuate nucleus and medial preoptic area, and with the kisspeptin antagonist peptide 234 into the lateral cerebral ventricle. The results of immunohistochemical staining revealed that pulsatile luteinizing hormone secretion was suppressed after injection of antagonist peptide 234 into the lateral cerebral ventricle, and a significant increase in luteinizing hormone level was observed after kisspeptin-10 injection into the arcuate nucleus and medial preoptic area. The results of an enzyme-linked immunosorbent assay showed that luteinizing hormone levels during the first hour of kisspeptin-10 infusion into the arcuate nucleus were significantly greater in the 100 pM kisspeptin-10 group than in the 10 pM kisspeptin-10 group. These findings indicate that kisspeptin directly promotes gonadotropin-releasing hormone secretion and luteinizing hormone release in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. The arcuate nucleus is a key component of the kisspeptin-G protein-coupled receptor 54 signaling pathway underlying regulating luteinizing hormone pulse secretion.

Key words: neural regeneration, basic research, gonadotropin-releasing hormone, enhanced green fluorescent protein, transgenic, luteinizing hormone, G protein-coupled receptor 54, medial preoptic area, arcuate nucleus, anteroventral periventricular nucleus, organum vasculosum of the lamina terminalis, photographs-containing paper, neuroregeneration

. Kisspeptin调节黄体生成素分泌的关键部位在弓状核[J]. 中国神经再生研究(英文版), 2013, 8(2): 162-168.

Haogang Xue, Chunying Yang, Xiaodong Ge, Weiqi Sun, Chun Li, Mingyu Qi. Kisspeptin regulates gonadotropin-releasing hormone secretion in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats[J]. Neural Regeneration Research, 2013, 8(2): 162-168.

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引言

材料方法

Design

A randomized, controlled animal experiment.

Time and setting

Experiments were performed in the School of Medicine Laboratory, Beihua University, China from October 2009 to October 2011.

Materials

All clean Wistar-Imamich rats obtained from Shanghai SLAC Laboratory Animal Co., Ltd., (License No. SCXK (Hu) 2010-008) were housed under controlled conditions (12-hour light/dark cycle with lights on at 19:00; temperature at 22 ± 2°C) and provided with free access to food and water. Rats ranged from 8 to 13 months of age, weighing 250–350 g, with equal numbers of male and female rats. The protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China^[22].

Methods

Preparation of GnRH/EGFP transgenic rats

Wistar-Imamich rat fertilized eggs were used for microinjection and embryo transfer was performed to establish GnRH/EGFP transgenic rats as follows. High-quality single-cell fertilized embryos in batches of 30–40 were placed under an Olympus inverted microscope for male pronuclear microinjection^[23-24]. To ensure maximum consistency, the impact of the needle was excluded. Injection needles of the same specifications and from the same batch were used. An IM-300 automatic injection device (Narishige, Sea Cliff, NY, USA) was used to control injection pressure and dose. After injection, embryos were transferred in drops of fresh M16 culture medium at 37°C, in a 5% CO₂ incubator, for 60 minutes. The numbers of living embryos were examined under an inverted microscope and recorded. Egg cytoplasm with a complete egg plasma membrane and a normal perivitelline space was taken as the indication for a living embryo. Those showing cytoplasmic diffusion were considered to represent embryonic death. Surviving single-cell embryos were bilaterally transplanted through the fallopian tubes of Wistar-Imamich pseudopregnant rats in the same estrus period, with 8–12 embryos being implanted on each side. At 19–22 days after transplantation, the number of births was recorded^[25].

Integration of PCR detection of the first established rats

DNA was extracted from rat tail tissue. A pair of specific primers was designed based on the microinjection sequence (Table 1). PCR was used to amplify a 278-bp DNA fragment (Figure 5), the presence of which indicated successful model establishment. Finally, a total of 50 GnRH/EGFP transgenic rats of both genders were obtained (aged 11.7 ± 0.6 months).

GnRH protein expression in rat brain tissues, as determined by immunohistochemical staining

Briefly, brain tissue sections were fixed for 1 hour in 4% paraformaldehyde (Sigma, Poole, UK), washed twice with PBS, and incubated for 1 hour in 2% (v/v) normal goat serum (Invitrogen, Paisley, Scotland) in PBS supplemented with 1% (w/v) bovine serum albumin (Invitrogen) and 0.3% (v/v) Triton X-100 (Invitrogen). Slices were then placed overnight in buffer containing mouse anti-human polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), which recognizes amino acids 6–10 of GnRH in pro-GnRH and GnRH, at a dilution of 1:10 000. On the following day, slices were washed twice with PBS supplemented with 0.3% bovine serum albumin and 0.1%Triton X-100, incubated for 1 hour in 7-amino-4-methylcoumarin-3-acetic acid- conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) at a dilution of 1:100 in buffer, washed twice with buffer, three times in PBS followed by H₂O, and then mounted on poly-L-lysine-coated glass slides using an aqueous medium. Immunostained slices were observed using an upright fluorescence microscope (Olympus BX51, excitation filter G365, dichroic mirror FT395, and emission filter LP420, Tokyo, Japan)^[26-27].

Kisspeptin or antagonist infusion into the lateral ventricle

On the morning of experiment, a single internal injection cannula with extension tubing, preloaded with the selective kisspeptin antagonist peptide 234 (7.5 nM in 12 μL of artificial cerebrospinal fluid) or artificial cerebrospinal fluid (12 μL) was inserted into the guide cannula, extending 1.0 mm beyond its tip to reach the left ventricle. After 2 hours of venous blood sampling at the elbow, kisspeptin antagonist peptide 234 or artificial cerebrospinal fluid was infused into the relevant animals over a period of 3 hours. Blood sampling continued throughout the experiment. Blood samples were frozen at –20°C for later assay to determine luteinizing hormone concentrations by ELISA^[26]. The method of injecting 1 pM, 10 pM and 100 pM kisspeptin-10 + 400 nL of artificial cerebrospinal fluid into the arcuate nucleus or medial preoptic area was as described above.

Intra-arcuate nucleus or intra-medial preoptic area infusion of kisspeptin-10

For administration of kisspeptin-10, bilateral injection cannulae (Plastics One) with extension tubing preloaded with different doses of kisspeptin-10 (Sigma, St. Louis, MO, USA) in 400 nL of artificial cerebrospinal fluid per brain locus (arcuate nucleus or medial preoptic area), or artificial cerebrospinal fluid, were inserted into the guide cannulae, extending 1.0 mm beyond its tip to reach the site of the brain nuclei. The distal end of the tubing was extended outside of the animal cage to allow remote infusion without disturbing the rats. The automated blood sampling system was set up in the same way as described above for the collection of 25-mL blood samples every 5 minutes, but only for 5 hours. All treatments were given by injection over 5 minutes after 2 hours of blood sampling.

Pulsatile GnRH release level, as detected by ELISA

In rats, a strong temporal relationship exists between the onset of a GnRH pulse in hypothalamic perfusates and the secretion of luteinizing hormone into the plasma, indicating that release of luteinizing hormone is a reasonable index of episodic GnRH release^[28]. Accordingly, we used repetitive sampling of venous blood in castrated rats to determine the pattern of luteinizing hormone secretion. On the following day, sequential blood samples were taken between 800 and 1 200, an interval during which episodic luteinizing hormone secretion has been demonstrated in rats. Blood samples were collected in 1-mL syringes at 12-minute intervals for 3 hours^[29-31]. The luteinizing hormone level was detected using an ELISA kit (Santa Cruz Biotechnology).

Statistical analysis

Data are expressed as mean ± SEM. Differences in luteinizing hormone pulse frequency were compared using one-way analysis of variance (α = 0.05). In the case of steroid-induced and mating-induced responses, differences in plasma luteinizing hormone concentrations were also analyzed by one-way analysis of variance (α = 0.05). Differences between two groups were analyzed utilizing paired samples t-tests (α = 0.05).

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