| In 1994, Zhang et al. cloned the structure of the mouse obese(ob) gene and its human homologue by positional cloning. The protein named leptin is the product of the ob gene. The discovery of leptin confirmed the hypothesis about forty years ago: energy stores represented by the adipose tissue might produce some sigalling molecules able to modulate both energy banlance and reproduction to CNS sites. In C57bl/6J ob/ob mice, the ob gene is mutated, resulting in a lack of functional leptin, and in db/db mice the mutated db gene resulting in a lack of leptin receptor. Both of them display a similar phenotype, with severe and early-onset obesity, extreme insulin resistance, hyperphagia, reduced energy expenditure and infertility in both sexs. However, intraperitoneal injections of leptin into ob/ob mouse decreased their food intale and body weight to normal, and also retrieved reproduction abibity. In femal ob/ob mouse, ovary and uterine weight growed, serum LH raised, primordial follicles and vesicular follicles increased. In male ob/ob mouse, testis and seminal vesicle growed, sperm increased. The observation suggested that leptin controled reproduction as modulating energy expenditure and food intake. Though the accumulated evidence suggests that leptin is important in control reproduction, whether leptin modulate hypothalamus-pituitary-gonadal axis directly or indirectly, the conclusion still need more research to clarity. Therefore, we initiated the studies of the possible effects of leptin on hypothalamus. We observed possible effects of leptin on NPY neuron expression in hypothalamus, studied the mechanism of action about leptin how to modulate reproduction, and provided some experiment data for leptin's clinical application. Results of the studies are as follows. 1 Coexpression of obR and NPY in mouse hypothalamus Objective: Leptin as a metabolic sigalling between nutrition and reproduction, is importan in controlling hypothalamus-pituitary-gonadal axis. Most experts observed that the GnRH neuron was not colocalized with obR and obRmRNA, so they presumed that leptin modulate indirectly GnRH neuron to effect its secretion. At present NPY in hypothalamus is maximal studied as an important modulator participating in energy balance and neuroendocrine, so we hypothesised that NPY would mediate the modulation between leptin and GnRH neuron. First of all, we observed the distribution of obR and NPY, and the coexpression of them in mouse hypothalamus. Methods: Adult femal C57bl/6J mice were perfused quickly with saline and 4% paraform through left ventricle. The brain tissue was cut between optic chiasma and mamillary body, and substitued fixation solution with gradient sucrose solution. We used immunohistochemistry to observe the distribution of obR and NPY, and studied the coexpression of obR and NPY with double immunohistochemistry in mouse hypothalamus. Results: 1 Results of obR positive cells: obR positive cells distributed as clump in hypothalamus ME, ARC and VMN, having obscure boundary. Cell membrane and body showed brown or yellow color. ObR positive cells were also present in choroid plexus, brain ependymal layer cell and vascular endothelial cell, showing strong positive staining. 2 The results of NPY positive neurons: in ARC of mouse hypothalamus, NPY positive neurons were present having bright red color in cell plasma. The NPY positive neurons are round or ellipse, having many neurites. NPY positive fibers were present in ME. A lot of NPY positive neurons and fibers were also present in cerebral cortex and hippocampus. 3 Coexpression of obR and NPY in hypothalamus: the result of double immunohistochemistry was as follows, obR positive cells were mainly present in ARC, ME, choroid plexus and so on, showing deep purple color. ObR positive cells distributed as clump having obscure boundary. Many NPY positive neurons were present in cerebral cortex and hippocampus, showing bright red color in cell plasma and having single or many neurites. In mouse hypothalamus ARC, brown purple or dark purple granula were presentin membrane and plasma of some NPY positive neurons, which were coloration material of obR antiserum. The coexpression of obR and NPY showed black color. Conclusions: ObR positive cells were present in ME, ARC, VMN of mouse hypothalamus, choroid plexus, brain ependymal layer cells and vascular endothelial cells. NPY positive neurons were present in ME, ARC, cerebral cortex and hippocampus and so on. In mouse hypothalamus ARC, coexpression of obR and NPY was present. 2 Effects on NPY gene expression by lateral ventricle administration of leptin in mice Objective: Most experts observed that the GnRH neuron was not colocalized with obR and obRmRNA, so they presumed that leptin modulate indirectly GnRH neuron to effect its secretion. From the anterior results, it had been known that the coexpression of obR and NPY were present in ARC of mouse hypothalamus. Combined the others experimental results, we considered that NPY neuron played an intermedial transmitter role. Therefore we began to study effects on the serum FSH and LH, at the same time, NPY gene expression in hypothalamus by lateral ventricle administration of leptin. So we could obtain that leptin effects GnRH neurons secretion by whether inhibiting or facilitating NPY gene expression. Through this part of experiment, we could studied the mechanism of action about leptin modulate GnRH neuron secretion, and provided some foundation experiment data for leptin's clinical application. Methods: Adult femal mice of the C57bl/6J strain were ovariectomized in sterile condition. After operation 10 days later, mice were injected s.c. with 2μg of estradiol benzoate in oil everyday and were used for 10 days. In the experiment, 4μg of leptin dissolved in 2μl saline was microinjected into the lateral ventricle in treated group and the equal volume of saline (2μl) was microinjected in control group. Blood samples and hypothalamus tissues were collected after microinjection 1.0h, 1.5h, 2.0h and 2.5h later respectively. According to the 《The Mouse Brain in stereotaxic coordinates》edited byGeorge Paxinos and Keith B. J. Franklin, lateral ventricle was located: passing through bregma, posterior distance 0.5 mm, lateral distance 1.0 mm, deep distance 2.0 mm. Blood samples were collected by removal mice eyes and stored at -20℃after centrifugation until measurements of FSH and LH were made by radioimmunoassay using kits. Hypothalamus tissues were collected immediately and weighted. Then, hypothalamus were put into 1ml of saline, grind into homogenate under lower temperature, centrifuged for 15 min at 4℃3000rpm. The supernatants were collected and stored at -20 ℃until measurement of NPY in mice hypothalamus were made by radioimmunoassay using kits. According to the Trizol interpretation, hypothalamus tissues were stored at liquid nitrogen and RNA was extracted strictly. After detection and quantitation, expression of NPY mRAN after microinjection was measured by RT-PCR. All the results were analysed by statistical analysis. Results: 1 Effects on mice serum FSH by lateral ventricle of leptin: after microinjection 1.0h later, there was no significent changes on serum FSH level in treated group and control group(P>0.05). But 2.0h later, compared with control group, serum FSH leve increased quickly in leptin-injected animals, and there was significant difference between the two groups(P<0.05). FSH level decreased slightly after microinjection 2.0h later, but there was still significant difference between control group and leptin-injected group(P<0.05). There was no significant difference between treated group and control group 2.5h later(P>0.05). 2 results of mice serum LH level by lateral ventricle of leptin: measurements of redioimmunoassay were as follows: after microinjection 1.0h later, serum LH level increased significantly, and there were significent changes in treated group and control group(P<0.05). 1.5h later compared with saline, serum LH leve increased continuously in leptin-injected animals. After microinjection 2.0h later serum LH level still was at a highly level, and there was significant difference between the two groups(P<0.05). However 2.5h later, mice serum LH level decreased in leptin-injected group, and there was no significant difference when compared with control group(P>0.05). 3 Effects on NPY gene expression by lateralventricle of leptin: 1.0h later after lateral ventricle injection, NPY expression in mice hypothalamus increased sharply and compared with control group, there was significant difference in leptin-injected group(P<0.05). Both 1.5h later and 2.0h later after microinjection, NPY expression in hypothalamus maintained at higher level constantly and also there was significant difference between leptin-injected group and control group(P < 0.05). 2.5h later, expression of NPY in hypothalamus decreased in leptin-injected group, and there was no significant difference between the groups(P>0.05). After microinjection 1.0h later NPYmRNA expression in mice hypothalamus increased also, there was significant difference in leptin-injected group when compared with control group(P < 0.05). NPY mRNA expression in hypothalamus increased in leptin-injected animals both 1.5h later and 2.0h later, and there was significant difference between control group and leptin-injected group (P<0.05). 2.5h later, there was no significant difference between control group and leptin-injected group(P>0.05). Conclusions:Lateral ventricle microinjection of leptin to OEP mice increased serum FSH concentrations with 1.5h and 2.0h later, but 2.5 h later plasma FSH level returned to level that there was no significant difference compared with control group. The injection of leptin increased serum LH leves with 1.0h later, and LH concentrations keeped at higher level until 2.0h later returned to level that there was no significant difference compared with control group. At the same time microinjection of leptin increased NPY gene expression in hypothalamus both protein level and transcript level. In OEP mice, NPY as an interneuron linking leptin and GnRH displays facilitation of GnRH neuron synthesis and secretion. 3 Effect of leptin on hypothalamus NPY neurons in vitro Objectives: The history of nervous tissue culture had more than 90 years from the initial tissue culture to present dissociated cell culture and single cell culture. Culture has many advantageous, for example it is easy to control experiment conditions and factors, and also easy to obtain alive results, moreover culture technique has been combined with many molecular biologytechniques together in experiment. Therefore culture is an effective technique in neuroendocrine research fields. Hypothalamus has so many neuron nucleuses and complex structures that it is difficult to study it in vivo. So many experts have constructed some culture techniques in vitro to study it. From the front results we have known that leptin could increase serum FSH and LH level in OEP mice, meanwhile increase NPY gene expression also in hypothalamus. In this part we could construct a good culture method of hypothalamus neurons, then observe effects of leptin on NPY gene expression in hypothalamus in vitro. Methods: Newly-born in 24h SD rats were used in this experiment. The hypothalamus was separated and digested into single cell suspension with 0.125% Trypsin 37℃for 30 min. The density of single cell suspension was adjusted into 5×106cell/ml with medium, and then was cultured on 6 well cell culture cluster smeared with PLL and LN in 36℃, 5% CO2 incubator. 24 h later the whole culture medium was replaced with serum-free defined medium. In the following days the serum-free defined medium was replaced 50% every 4 day. The growth conditions of hypothalamus neurons were observed with phrase contrast microscope everyday. When cultured for 3, 7, 10 and 14 day, we measured and analyzed the long diameter, short diameter and average length of neurits of neurons in vitro. Meanwhile Using NSE, NF,NPY antiserum to identify neurons and detect expression of neurons. The cell body and neurits of NSE, NF, NPY positive neuron were analysis by statistics with image analyses system. According to the results, we made sure the best growth period of neurons. Then we observed the effect of different concentration of leptin on NPY gene expression with radioimmunoassay and RT-PCR. Results: 1 At first, hypothalamus neurons from newly born rats were round and having bright halation. 24h later most of cells adhered, and cell body was round or oval-shape. A few cells that did not adhere were floating and died gradually. 36h later, some neurons extended one or two neuritis, and cell body was becoming from round to oval-shape or triangle. When culturedfor 3 days, some cells polymerized again, and extended long neuritis. Cell body growed up, and neurits growed in prosperity and had many branched graduallly. At the extrem of neurit there often was growth cone. After culture 5 day later, cell body and neurits of neurons went on increasing, and nucleus could be observed clearly. When cultured for 7 day, the neurits of neurons connected with each other just like a web, and the growth reached a peak. 10 and 14 days later cell body and neurits basically maintain a week condition. At same time, during the culture we could observe small amounts glial cell. With the growth of neurons, glial cells were cleavaging and increasing. Cell body of glia cells was irregular. With the development of culture time, glial cells and neurons formed connection. 2 Hypothalamus neurons cultured in vitro showed NSE and NF positive immunological reaction. NSE expressed in cell body and neurits, and NF expressed in cytoplasma and neurits. Both NSE and NF positive cells were round, oval-shape or irregular. The positive cells were dipolar or multipolar neuron. The positive rate of neurons under microscope was 78% 85%. The statistic results showed that with the development of culture time NSE and NF positive neurons growed and reached a steady state one week later. 3 Hypothalamus neurons could show NPY positive immunological reaction. NPY positive neurons were round or irregular. The cytoplasma and neurits showed brown or brown yellow color. During culture cell body of NPY positive neurons was growing up and neurits were increasing. When cultured for 7-10 day NPY positive neurons reached to a steady state. Then cell body and neurits of NPY positive neurons shrinked back gradually. 4 Compared with control group, with Leptin 10-10mol/l expression of NPYmRNA had no statistical difference, but with 10-8 mol/l,10-6 mol/l expression of NPYmRNA enhanced and had statistical difference. 5 Expression of NPY increased when given leptin with 10-8 mol/l,10-6 mol/l, and there was significant difference compared with control group. However when given leptin with leptin10-10mol/l there was no significant difference in NPY gene expression compared with control group. In medium compared with control group, NPY gene expression increased in leptin with 10-8 mol/l,...
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