Effects Of Photoperiod On The Hypothalamic-Pituitary-Gonad Axis Of Mesocricetus Auratus

ObjectivesTo investigate the effects of photoperiod on related factors in peripheral gonadal as well as the mRNA expression of gonadotropin-releasing hormone, gonadotropin-inhibiting hormone and its receptor in hypothalamic-pituitary-gonadal axis.To demonstrate that TCM syndrome caused by illumination changes is in harmony with ’hyperactivity of fire due to yin deficiency’ and to provide animal models mimicking precocious puberty for further study. Golden hamsters with obvious circadian rhythm are used for the study.MethodsFour nests of2-day-old female golden hamsters were divided into four groups, they were group N (12h,50lux), group Ml (16h,501ux), group M2(16h,350lux) and group M3(20h,3501ux),each group consisting of10pups and one maternal golden hamster. Male golden hamsters were the same as the female ones.All pups were weaned at21-day-old and then fed separately in a specially designed squirrel-cage. Artificial light was took in this study instead of sunlight until the pups were28-day-old. Light intensity in group M2and M3were controlled in3501ux while group N and M1were in50lux. Each group was set by one timer. The photoperiod for group N was12light/12dark while group M1and group M2were16light/8dark and group M3was20light/4dark. The mRNA expressions for hypothalamic GnRH, GnIH and Mellc genes were detected with Real-time PCR as well as GnRHR(GPR54) and GnIHR(GPR147) genes in the pituitary gland, so for GPR54in the uterus and ovary. SPSS13.0was applied for the statistical analysis and the results were displayed as mean±standard deviation (x±S).ResultsPart Ⅰ The impact of photoperiod on related factors in peripheral gonadal in female golden hamsters and male golden hamstersFemale:(1) Golden hamsters in group M1, M2and M3showed that they were more obvious restless, irritable than group N. Food and water intake were decreased in group M with scarce animal hair, compared with group N. The body weight gain for golden hamsters in group M3was significantly higher than that in group N (P<0.01) but body weight gain in group M1and group M2was significantly lost compared with that in group N (P<0.01).(2) Ovarian coefficient for golden hamsters in group M1(7.15±0.8) was significantly higher than the level in group N(5.72±2.2) with a p-value of0.07. Compared to the group N, the uterine wet weight, uterus coefficient and ovarian wet weight for group M1were off no significant differences. The uterine wet weight, ovarian wet weight and uterine coefficient of group M2were significantly lower than that in group M1(P<0.05) except for ovarian coefficient. There were no statistically significant differences between group M2and group M3in uterus wet weight, ovarian wet weight and uterine coefficient except for uterine coefficient.(3) The E2level in group N (95.25±25.79pg/ml) was significantly higher than those in experimental groups, but for group M1was significantly lower than that in group N (P=0.002). E2was not significantly different between group M2and group M1as well as between group M3and group M2. There was no significant difference about FSH and LH in each group.(4)There were no significant difference for thickness of endometrial epithelium between group M1and group N. So were endometrium, myometrium and the largest follicle diameter. Compared with corresponding parameters in group M1, the p-values of endometrial epithelium thickness (86.19±7.15μm), endometrial thickness (229.88±16.87μm), mometrial thickness (113.86±10.40μm), the largest follicle diameter (260.95±22.40μm) in group M2were0.884,0.278,0.157,0.325separately. There were no significant differences between group M3and group M2except for the largest follicle diameter with a p-value of0.039.Male:(1) The appearance and activities for male golden hamsters in group M1, M2and M3were the same as that for the female ones. The body weight gain for golden hamsters in group M1(17.63±4.07g),group M2(14.23±2.06g),group M3(19.07±3.43g) were of statistical significance compared with that in group N(26.71±3.09g), with a p-value of0.001.(2) There were no statistical significance for testicular wet weight and testicular coefficient among group N, group M1and group M2.Compared with the T,FSH and LH levels in group N (0.79±0.07ng/ml,21.75±0.55mIU/ml,51.19±0.82mIU/ml), the p-values in group M1(1.21±0.24ng/ml,21.41±1.22mIU/ml,52.86±2.07mIU/ml) were0.031,0.289and0.084separately. Compared with corresponding parameters between group M1and M2(1.14±0.13ng/ml,20.56±0.80mIU/ml,51.37±3.13mIU/ml), the p-values were0.529,0.276and0.152separately. The p-values were0.652,0.73and0.656, comparing those between group M2and M3(1.08±0.11ng/ml,21.31±0.35mIU/ml,50.84±3.48mIU/ml)(3) The HE testicular in group N presented that testicular seminiferous epithelium was in good shape and spermatogenic cells in different developmental stages were in good ordered arrangement. Correspondingly they were spermatogonia, primary spermatocytes, secondary spermatocytes, sperm cells, unmature sperm cells. In experimental group, the basic form of testicular seminiferous epithelium was in good shape and there were no pathological damage. No cells had grown into mature sperm.Part II The effects of photoperiod on mRNA expression of GnRH, GnIH and their receptors in the hypothalamic-pituitary-gonadal axis for golden hamstersFemale:(1) The mRNA levels of hypothalamic GnRH, GnIH and Mellc in group N were0.55±0.22,4.48±1.14and0.45±0.08separately. In compare with the corresponding parameters in group M1(0.17±0.05,10.68±3.11,0.15±0.03), the p-values were0.217,0.121and0.004.Compareing those parameters between group M1and group M2(0.28±0.03,11.92±0.69,0.19±0.05), the p-values were0.741,0.139and0.530.(2) Comparing the mRNA levels of pituitary GPR54and GPR147between group N(171.69±45.1,1.96±1.21) and group M1(237.13±34.3,0.084±0.038), the p-values were0.142and0.366. In compare with the corresponding parameters between group M1and group M2(90.4±26.3,0.55±0.15), the p-values were0.010and0.146.(3) The p-values were0.149and0.046, comparing uterine GPR54mRNA and ovarian GPR54mRNA in group M1(1.24±0.69,2.33±0.98) with those in group M2(0.24±0.07,0.23±0.07).Male:(1) The mRNA levels of hypothalamic GnRH, GnIH and Mellc in group N were0.42±0.17,0.70±0.15and0.99±0.56,compared with the corresponding parameters in group M1(0.37±0.13,8.95±3.02,0.19±0.09) the p-values were0.73,0.019and0.005separately. Comparing the corresponding parameters between group M1and group M2(0.11±0.02,1.15±0.18and0.19±0.07), the p-values were0.027,0.022and0.99. Comparing those parameters between group M2and group M3(0.19±0.04,7.99±2.06and0.13±0.04), the p-values were0.416,0.794and0.70. (2) Compared the mRNA levels of pituitary GPR54and GPR147in group N(254.46±52.78,8.83±3.09), with group M1(140.57±21.47,0.63±0.11) the p-values were0.052and0.004. Comparing the corresponding parameters between group M1and group M2(191.17±28.68,0.81±0.26), the p-values were0.39and0.941. The p-values were0.971and0.876, comparing the mRNA levels of pituitary GPR54and GPR147in group M2(191.17±28.68,0.81±0.26) with those in group M3(193.29±41.21,1.18±0.64).Conclusions(1) Female golden hamsters with the syndrome of "hyperactivity of fire due to yin deficiency" were successfully induced by extending the photoperiod. So were the male golden hamsters.(2) Photoperiod changes stimulated the basic metabolism of the golden hamster, but reduced food and water intake. As a result, the body weight of weaned hamsters alleviated.(3) As it is shown that long photoperiod is of multilevel inhibitory action on the HPG axis from lactation to delectation. It is supported by some pubertal maturation parameters (such as uteri and ovarian weight, vaginal opening) and sex hormones. The findings in our current data are that long photoperiod can down-regulate hypothalamic Mellc and GnRH mRNA expressions and increase GnIH mRNA expression significantly. One of the findings is that long photoperiod also down-regulate GPR54, GPR147mRNA expressions in the pituitary gland as well as GPR54mRNA expression in the uterus and ovaries. Another interesting phenomenon discovered in this study is that long photoperiod stimulate the development of ovarian follicles and the rise of testosterone level. it suggests that ovarian follicles and testosterone level for hamsters may be more sensitive to light exposure.