| Neurotrophin 4 (NT4) is multifunctional and plays extensive biological roles. The evidences show that neurotrophins express in reproductive tissues and regulate the follicular development, ovulation and hormones synthesis in ovary by paracrine and autocrine ways. Gonadotropic hormones are very important to animal reproductive regulation. Follicle-stimulating hormone (FSH) promotes the growth and development of ovarian follicles, proliferation of ovarian granular cells etc. LH accelerates the speed of blood flow, and cooperates with FSH, they induce follicle ovulation and promote corpus luteum generation. But there is little research on expressions of NT4 and TrkB in female animal reproductive system, and that interactions with gonadotropic hormones and their receptors was not reported. Our studies lay the foundation to clarify the biological functions and mechanisms of NT4 and TrkB in female animal reproductive activities. Adult cows were chosen as experimental subjects, and the main results are as follows:1. The location and quantitation of NT4 and TrkB in uterus, fallopian tube and ovary during luteal phase and follicular phase in cowStudy on the expressions of NT4 and its receptor TrkB in ovary, oviduct and uterus during follicular and luteal phase in cow. The results of IF showed that NT4 and TrkB expressed in preantral follicles of ovary, granular and endometrial cells of antral follicles in follicular phase and follicular, luteal cells of ovary in luteal phase. NT4 and TrkB expressed epithelial cells of uterine glands and uterine cavity, and epithelial cells of tubal mucosa during follicular phase and luteal phase. Through comparison, we found that the expression level of NT4 mRNA in ovarian tissue and that of TrkB mRNA in uterine tube tissue during luteal phase were significantly higher than the expression levels during follicular phase (p<0.01). During luteal phase, the expression level of NT4 mRNA in ovary was significantly higher than in uterine and oviduct (p<0.01). The expression level of TrkB mRNA in oviduct was significantly higher than in ovary and uterine (p<0.01). During follicular phase, there was no significant difference in the expression levels of NT4 and TrkB mRNA among different tissues. The results of western blotting also proved NT4 and TrkB both expressed in ovary, oviduct and uterine during follicular and luteal phase. The results showed that NT4 and TrkB had certain effects to the development of female animal reproductive system.2. The effects of gonadotropic hormones to the expressions of NT4 and TrkB in granular cells of ovaryStudy on the effects of LH and FSH to the expressions of NT4 and TrkB in granular cells of ovary. The results of fqPCR showed that the expression levels of NT4 mRNA were increased after adding LH. There was significant difference in the expression levels of NT4 mRNA were at 8 and 16 hrs,10 and 20μg/mL; 24 hrs,20μg/mL (p<0.05), but there was only significant difference in that of TrkB mRNA was in 40μg/mL with different treating times (p<0.05).Among the significant expression group. NT4 mRNA expression was best at 8 hrs,10μg/mL; TrkB mRNA expression was best at 8 hrs,40μg/mL. The expression levels of NT4 mRNA was best at 20μg/mL of 8,16 and 24hrs after adding FSH (p<0.05), the expression levels of TrkB mRNA were increased at 10 and 20μg/mL with different treating times (p<0.05). Among the significant expression group. NT4 mRNA expression was best at 8 hrs,20μg/mL; TrkB mRNA expression was best at 8 hrs,10μg/mL.The results of ELISA showed that there was no significant difference in NT4 protein expression levels after adding LH and FSH (P>0.05), NT4 protein was best at 8 hrs,20μg/mL by comparison, and proved that gonadotropic hormones had certain effects to the expressions of NT4 and TrkB.3. The effects of NT4/TrkB pathway to gonadotropic hormone receptors in granular cells of ovaryStudy on the effects of NT4 to the expression of gonadotropic hormone receptors in granular cells of ovary. The fqPCR results showed that the expression levels of LHR mRNA were increased after adding NT4. There was significant difference in the expression levels of LHR mRNA at 8.16 and 24 hrs,30μg/L group (p<0.05). among the significant expression group. LHR mRNA expression was best at 8 hrs.30μg/mL. After adding NT4. the expression levels of FSHR mRNA were also increased extremely significant difference at 8,16, and 24 hrs,60 and 100μg/L group (p<0.01). and there was significant difference at 16 hrs,30μg/L group (p<0.05), among the significant expression group. FSHR mRNA expression was best at 16hrs,100μg/mL., The expression levels of LHR mRNA decreased after adding K252a. The levels of 8, 16. and 24 hrs, 10μg/L group were significantly lower than that of control group (p<0.05). and other treated groups were extremely lower (p<0.01). The expression levels of FSHR mRNA also extremely decreased (p<0.01). The expression of LHR and FSHR were inhibited significant at 8 hrs,10μg/L by adding K252a. The results showed that NT4 could affect gonadotropic hormone receptors through the combination with its receptor, and the granular cells of ovary may function by the means of the interactions between NT4/TrkB and LH/LHR or NT4/TrkB and FSH/FSHR.In conclusion, our studies proved that NT4 and its receptor TrkB expressed in the cow reproductive tissues of different oestrous cycle and take part in regulate reproduction. NT4 has a time-dosage dependent to gonadotropic hormones in the granular cells of ovary. NT4/TrkB signal pathway has correlated to the expression regulations of gonadotropic hormone receptors in granular cells of ovary. |
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