| Animal reproductive physiology is the basis of learning and grasping animal reproductive rules. The regulation mechanism of the synthesis and the secretion of animal reproductive hormone is the focal point of the research on animal reproductive physiology. In the hypothalamus-hypophysis-gonad axis, hypophysis lies in the central place. Prehypophyseal gonadotroph synthesize and secrete FSH and LH, which are combined to be called GTH. The secretary condition of GTH directly decides animal empathema phrase, on the other hand, the synthesis and the secretion of GTH is influenced by many factors including mainly GnRH secreted by hypothalamus and steroid hormone secreted by gonad. The positive and negative feedback regulations of various factors ensure the balance of the reproductive endocrine system in animal bodies so that animals can maintain normal reproductive activities such as empathema, androlepsis and accouchement.As for the regulation mechanism of the secretion of GTH, the mechanism before receptor is clearly studied. For the moment the hot research spot focuses on the mechanism after receptor. By comparison, this research is less fruitful and many questions still remain to be answered.This thesis attempts to explore the post-receptor signal transduction mechanism of the synthesis and secretion of mammal GTH, which is not known clearly by far. We will observe the response of the signal transduction pathway of the pituicyte of original generation of rats cultured in vitro while being stimulated by GnRH at different impulse frequencies, especially the PKC pathway and the cAMP pathway. We will also quantitively detect the level change of PKC and cAMP in cells while being stimulated by GnRH at different impulse frequencies, at the same time we will quantitively detect the expression amount of LHβmRNA, FSHβmRNA, LH and FSH. In this way we can observe the different responses of these two signal transduction pathways in cells to the stimulation of GnRH at different impulse frequencies. This research aims to further explain at molecular level the post-receptor regulatory mechanism of the synthesis and the secretion of GTH. It consists of four experiments.The first experiment is called"The Observation of the Primary Culture in Vitro of Rats'Adenopituicytes and Their Secretion of GTH". It aimed to establish the way of the primary culture in vitro of rats'adenopituicytes, to observe the living rules of primary pituicytes and determine the most athletic functioning period of GTH cells in pituicytes primarily cultured in vitro so as to make preparations for further experiment. During this experiment, the hypophysis of 4-month-old female albino rat during Rut, after being digested by collagenase, was inoculated and cultured in the 96-well cell cluster flat bottom. On the 1st day, while being inoculated, the cells were scattering, with each individual cell floats alone on the culture fluid, while on the 2nd day, the cells gathered, adhered to each other and formed an island, with few of them stayed alone; on the 3rd day, almost all cells adhered to each other, with a few of them stretched out their parapodium and began to grow rapidly; on the 4th and 5th day, the cells extended fully into Fusiform shapes and grew prosperously; on the 6th and 7th day, the cells grew most prosperously. They joined into one piece and filled the entire well, with endochylema turgor vitalis, cytoplasm transparent and the edge sharp; on the 8th day, although they still filled the well, the cells appeared a little aged and their morphous began to change and showed a tendency of fibrosis. The detection of the secretory volume of FSH and LH shows that the secretory volume is rising day by day except that the volume on the 3rd day is higher than that on the 4th day. It reaches the top on the 7th day and reduces gradually since then. From the perspective of the immunohistochemical stain, from the 4th day to the 7th day, the masculine rate of GTH is rising gradually and it reaches the top on the 7th day, which shows that during this period, the GTH cells are restoring from the damaged state to the normal state. While on the 8th day, the masculine rate of FSH and LH begin to fall, which shows that the GTH cells begin to show fibrosis so the masculine cells are decreasing. Thus from the 3 angles the morphous, the hormone secretory volume and the masculine rate of cells, we can draw a coincident conclusion that the most prosperous growing period of rats'pituicyte cultured in vitro is the 7th day. The second experiment is called"The Analysis of the Influence of GnRH Impulse mode on the Secretion of GTH and the Response Feature of LH/FSH". It aimed to research the influence of GnRH impulse mode on the secretion of GTH and analyze the response feature of LH/FSH. In the primary stimulation experiment of GnRH at different frequencies, we devised 4 impulse frequencies, which were respectively at the intervals of 30min, 60min, 120min and 240min. At each frequency, we devised different amplitude of vibration, the height of which (the concentration of GnRH) was respectively 0, 0.1, 1.0, 10, 100, 500 and 1000nM and the width of which (the functioning duration of GnRH ) was respectively 10, 20 and 30min. The stimulation of each type of impulse lasted 24h, and then the corresponding secretory volume of FSH and LH was detected. The result shows that under the condition of this laboratory, the most suitable height of GnRH pulse amplitude (the concentration of GnRH) for the response of FSH和LH is within 100nM, while the most suitable width (the functioning duration of GnRH ) is 30min. On this basis, during the formal stimulation experiment of GnRH at different frequencies we determined the functioning duration (the width of the vibration amplitude) of GnRH as 30min, and the functioning concentration (the height of the vibration amplitude) as 5.0, 20, 50 and 80nM. On this basis, we devised 4 impulse frequencies, which were respectively at the intervals of 30min, 60min, 120min and 240min. The result shows that in 24 hours the secretory volume of LH reaches the top when the frequency of GnRH impulse is 30min and the vibration amplitude of it is 20nM, moreover, with the stepping down of the frequency of GnRH stimulation, the secretion of LH is reducing gradually. In 24 hours the secretory volume of FSH reaches the top when the frequency of GnRH impulse is 120min and the vibration amplitude of it is 20nM, moreover, with the hurrying up or the stepping down of the frequency of GnRH stimulation, the secretion of FSH is both reducing gradually. The stimulation of GnRH in the other 3 vibration amplitude groups has the same tendency. The result of this experiment shows that GnRH impulse frequency itself is a regulation signal for the GnRH of different impulse frequencies has clearly different influence on the expression of LH and FSH. In the condition of the same vibration amplitude, when the cells are stimulated by impulse at high frequency (at the interval of 30min ), the secretion of LH reaches the top, while when they are stimulated by impulse at low frequency (at the interval of 120min ), the secretion of FSH reaches the top.The third experiment is called"The Influence of two GnRH Impulse Modes on the Expression of FSHβmRNA and LHβmRNA in Rats'GTH Cells Primarily Cultured in Vitro". By applying the technique of culture in vitro of cells in combination with the fluorescent quantitation of PCR, this experiment aims to research the change of FSHβmRNA and LHβmRNA in cells when the pituicytes of original generation of rats cultured in vitro were stimulated by GnRH at different impulse frequencies, to affirm the different response of GTH subunit gene expression to the stimulation of GnRH at different impulse frequencies and to observe especially the expression of FSHβmRNA and LHβmRNA when the impulse frequencies were 30min and 120min. It further explains at molecular level the influence of GnRH at different impulse frequencies on the secretion of LH and FSH by GTH. The result shows that GnRH at different impulse frequencies has the influence on the expression of GTH mRNA. In the condition of the same vibration amplitude, when the cells are stimulated by impulse at high frequency (at the interval of 30min ), the level of LHβmRNA reaches the top, while when they are stimulated by impulse at low frequency (at the interval of 120min ), the level of FSHβmRNA reaches the top. This further supports the hypothesis that GnRH impulse frequency itself is a regulation signal.The forth experiment is called"The Influence of Excited or Inhibited PKC or cAMP in Cells on the Expression of GTH Being Stimulated by GnRH at Different Impulse Frequencies". This experiment, on the basis of previous ones, aimed to observe the response feature of LH and FSH to the stimulation of GnRH at different impulse frequencies after PKC or cAMP in cells were excited or inhibited so as to further analyze the post-receptor signal transduction mechanism of the expression of LH/FSH. The activator and inhibitor of cAMP were respectively FSK and SO22,536, the activator and inhibitor of PKC were respectively PMA and H7. The result of the experiment shows that FSK and SO22,536 can significantly improve or reduce the content of cAMP in GTH cells but do not influence the activity of PKC, while PMA and H7 can significantly enhance or weaken the activity of PKC but do not influence the content of cAMP; when the use of the activator and the inhibitor of cAMP makes the cAMP level rise or fall, the expression of LH is influenced significantly, that is to say, the level of LH rises with the rise of cAMP and falls with the falling of cAMP, while the influence on the expression of FSH is not striking; when the use of the activator and the inhibitor of PKC makes the PKC level rise or fall, not only the expression of LH but also the expression of FSH is influenced significantly. The level of both rises with the rise of PKC and falls with the falling of PKC.This thesis researched the response feature of LH and FSH in the pituicyte of rats primarily cultured in vitro while being stimulated by GnRH at different impulse frequencies and compared the post-receptor signal transduction pathway. The result shows that GnRH impulse frequency itself is a regulation signal. When the cells are stimulated by GnRH impulse at high frequency, the post-receptor signal transduction pathway is cAMP-PKA and PKC-Ca2+ and it brings about mainly the response of LHβ; when they are stimulated by GnRH impulse at low frequency, the post-receptor signal transduction pathway is PKC-Ca2+ and it brings about mainly the response of FSHβ.
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