| Animal reproductive physiology is the base of understanding and controlling the regularity of animal reproduction. The focus of this research is on animal reproduction, especially the regulatory mechanism of the synthesis and secretion of animal reproductive hormones. In hypothalamus-pituitary-gonads, pituitary is at the center position. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are biosynthetized and secreted by prehypophysial gonadotroph. Both of them are called gonadotrophic hormone (GTH). The animal oestrus is decided by the secretion of GTH. The synthesis and release of these two pituitary glycoproteins are controlled by the complex interaction of multiple factors. The most important one is GnRH secreted by hypothalamus, and other steroid hormones such as estrogen, progestogen and androgen. The factors mentioned above have the effect on the secretion of GTH through positive or negative feedback. There exist other factors confirmed in the regulation of the biosynthesis and secretion of GTH, such as growth hormone (GH), gonadotrophic releasing inhibiting factor (GRIF), insulin-like growth factor (IGF), activin and inhibin. Owing to the factors, the secretion of GTH can balance the endocrine system at the right time, in the right dosage and at the right pattern, so the animal can keep the normal reproductive activities, such as oestrus, pregnancy and parturition.There have been many researches on the regulatory mechanism of the GTH secretion pre-receptors. So we focus on the mechanism post-receptors of mammal GTH secretion. Generally speaking, there are fewer researchers and fewer papers, and a lot of problems have not been worked out, compared with the research of pre-receptors. Recently some channels that are related to the signal transduction of post-receptors have been found. And some hypotheses have been referred. It has been indicated in hard bone fish that the expression of differential GTH subunits has different channels of signal transduction. But it's not confirmed in mammal, especially in vitro. What kind of signal channel is not clear in the culture of mammal pituitary cells after varying GnRH pulse frequency. The purpose of this research is:1) to figure out the mechanism, which has not been clear yet, of mammal GTH synthesis and secretion post-receptors;2) to observe the signal channel will make what kind of response after varying GnRH pulse frequency and the expression of LHβmRNA, FSHβmRNA, LH and FSH detected at the real time;3) to make it confirmed that the response of the signal channel after varying GnRH pulse frequency and the effect of the expression of the GTH subunit. The result of this research will help people get further insight of the regulatory mechanism of mammal GTH biosynthesis and secretion, and comprehend the discipline of mammal reproduction and artificial regulation.The function of FSH is to activate stationary ovary, induce estrus. The function of LH is ovulating and forming corepus luteum. Both of them have two subunits. One is common termedαsubunit. The other one is specific termedβsubunit, which is the difference of the two hormones. Accordingly, to detect FSH or LH specifically is to detect theβsubunits or their mRNAs. According to the gene orders, the specific primers and probes were designed conveniently. By using the technique of real-time PCR, FSHβmRNA or LHβmRNA is quantitated.To provide direct evidence of differential regulation of GTHβsubunit mRNA expression and GTH secretion by varying GnRH pulse frequencies at the level of pituitary cells, we used an in vitro serum-free culture model of primary rat pituitary cells. According to the result of the preliminary experiment, primary monolayer cultures of rat pituitary cells were treated with 100nmol/L GnRH (10 min/pulse) pulses for 24h at a frequency of every 30, 60, 120 or 0min. Total RNA was prepared from cultured cells based on the same numbers, and then the reverse transcription. cDNAs from each groups were prepared for FSHβmRNA or LHβmRNA quantitated.Analyzed by SPSS 13.0, the result shows that pulsatile GnRH significantly stimulated FSHβmRNA and LHβmRNA levels at all pulse frequencies tested. LHβmRNA levels were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min (P<0.01). In contrast, FSHβmRNA levels were stimulated to the greatest extent at lower GnRH pulse frequency, such as every 120 min (P<0.01).Based on all the information above, we demonstrate differential effects of varying GnRH pulse frequency in vitro in primary monolayer cultures of rat pituitary cells. The peak of FSHβmRNA or LHβmRNA appeared after varying GnRH pulse frequency. The patterns of signal transduction into cells might be through different signal channels after GnRH binds to its receptors, and the function of cells might change. The different patterns might cause the different synthesis and secretion of GTH. The result of this research provided good beginning for the continue studies.
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