| Circadian rhythms are driven by cellular oscillators known as clocks or pacemakers and are an important regulator of a range of physiological and behavioral processes. Circadian clocks are entrained to local time by daily light-dark cycles, a mechanism that endows them with the ability to anticipate and adapt to daily seasonal changes in environmental modalities. Many birds exhibit a diurnal rhythm in ovulation-oviposition cycle, such as, the ovulation always take place behind the last oviposition, the time preference of ovulation, and the pause day after a clutch. The chronobiology and reproductive endocrinology has enabled us to understand and manipulate vertebrate reproduction from an entirely new perspective. However, it's a complex regulatory network involved in several neuronal and hormonal pathways within the hypothalamic-pituitary-gonadal axis. The researches in neuroendocrine, development of follicles and the egg formation can not explain the whole process of ovulation-oviposition well. In this study, we tried to understand the rhythmicity during the ovulation-oviposition cycle by exploring the circadian clock system. 1. The record and analysis of rhythmic egg-laying patternWe monitored the daily laying patterns of each individual by checking the oviposition time at 30-min intervals for some reproductive parameters, such as:the distribution of egg-laying time, the interval between two ovipostion, the length of a clutch, and the character of pause day. We found that the time preference of hens'oviposition was very apparent, oviposition occurs mainly during the 8:00 am-12:00 am. The high performance hen laied an egg at 24 hour intervals. On the day before the paused day, the time of oviposition mainly distributed in morning and evening. So the study confirmed the "open period" of ovulation (ovipostion) got the consistent conclusion with former studies.After 4 wk observation, we selected enough birds which have consistant laying sequences and ovipostion time for further experiments.2. The rhythmic expression of clock genes and the location of circadian clockworkBased on the molecular bases of circadian clock, we can identify whether the circaian clock exist in some tissue or cell by measure and overlay the expression pattern of clock genes (Bmall, Bmal2, Clock, Per2, Per5, Cry1, Cry2). When the functional clock run, the clock gene should express rhythmic like the cosine curve, and had specific acrophase pattern. We quantified the expression pattern of seven core clock genes in granulosa and theca cell of pre-ovulatory follicles and four segments of oviduct. After the cosinor analysis, we confirmed the the functional clock exsist in the granulosa cell of F1-F3 follicles, the infundibulum and the uterus but not in other tissues which we measured. And in F1, the amplitude of oscillator is the most powerful, F2 is next, F3 is the weakest. But they have the consistent arophase (the peak time of marker clock gene). When compare the acrophase between the infundibulum and the uterus, the uterus precedes significantly than infundibulum. Our study provides the molecular explanation for those rhythmic events in gonadal hormone secretion and egg formation.3. The regulatory mechanism of LH induce the clock genes' expressionAfter added the signal pathway inhibitor, we explore the mechanism of LH-induced pathway by quantify the clock genes' expression. We set the Dexamethasone-treated granulosa cells as the control group, and add LH in the experimental group. The results show that the clock gene mRNA expressed smoothly regardless in control and experimental group, and without significantly cosinor pattern. The LH treatment can induce significantly the expression level of Bmal1 at ZT4, whereas enhanced Per2 expression in ZT4 and ZT8. There is not any oscillation in Clock expression, and the significant increase of Cryl expression are found on ZT12,20,24. The cAMP inhibitor can block the LH stimulated effects in Bmal1 and Per2, suggested that the reliance on cAMP signal pathway. The effects of LH on Bmal1 expression not diminished significantly by adding individual Erkl/2 or p38 MAPK inhibitor, but got the inverted results in Per2. However, the cooperatively inhibit of Erk1/2 and p38 MAPK pathway, the expression of Bmall and Per2 are closed to the control group and significantly lowed than LH-treatment group. This results indicated that Erkl/2 and p38 MAPK cooperate with each other to transmit the LH stimulous signal to clock genes.4. The screening of clock controlled genes in the F1 follicles granulosa cells by RNA-seqWe used the RNA-seq to study the trancriptome changes betweeen 24 h, and got the rhythmic expressed genes that are directly or indirectly clock controlled genes by using the JTK_CYCLE package in R project. We aimed to enrich the regulatory pathways involved with the ovarian clock.We got 365 genes that showed rhythmic expression pattern, about 5% of the total input genes, further, we performed the Gene Ontology (GO) annotation and KEGG pathway enrichment. Our results showed that the cyclic expressed enriched significantly to two pathways, ribosomal protein and spliceosome protein. The ribosomal proteins abundance and phosphorylated state are tightly linked to the tumorigenesis. The resluts suggested that the hen appealing as a model for the ovarian cancer research. The splicesome protein mainly take part into the pre-mRNA splicing progress. It's an important post-transcriptional modification which can affect the abundance of functional transcript. This finding demonstrated that the core clockwork can disrupt the downstram gene expression by post-transcriptional modification. In this manner, the splicesome amplify the circadian signal to the whole transcriptome.This study integrated the evidences of circadian system by the measurement of clock genes expression and the analysis of rhythmic character, to locate the clockwork in the hens'reproductive system, explore the pathwaybetween the LH and some clock genes, and screening the possible clock-controlled genes in the F1 granulosa cells, which was established a big picture of chronobiology in ovulatory-oviposition cycle. |
PDF Full Text Request