| In order to adapt to the needs of the natural selection,animals form a breeding phenomenon that occurs only in particular breeding seasons;this phenomenon is the seasonal reproduction.There are many factors affecting the seasonal reproduction of animals,and photoperiod is a typical seasonal factor that regulates reproduction in seasonal breeders.The regulatory mechanism of photoperiod on seasonal reproduction is a complex process involving various regulatory factors.Opsin5(OPN5)is an important member of non-visual opsin which is considered to be deep-brain photoreceptors involved in the seasonal reproduction of animals.However,the genetic mechanism that OPN5 in regulating seasonal reproduction is not yet clear.Cricetulus barabensis is widely distributed in farmland in northern China with a high reproductive capacity in spring(March to April)and autumn(August to September).C.barabensis is sensitive to photoperiod and is an ideal laboratory animal for studying the photoperiodic regulation of seasonal reproduction in animals.In order to reveal the possible role of OPN5 in the regulation of animal reproduction,we investigate the expression pattern of OPN5 using molecular biology and immunohistochemistry in this study.Firstly,the cDNA sequence of OPN5 was cloned by RT-PCR from C.barabensis.The sequence is 1340 bp in length,containing part of the 5' noncoding region,part of the 3' noncoding region and a complete open reading frame of 1134 bp which encodes a polypeptide of 377 amino acids residues(KY949483).The content of G+C is higher than A+T in the sequence,indicating that its Tm value is higher and the thermal stability is strong.The analysis of amino acids composition,transmembrane region and hydrophobicity suggests that OPN5 contains N-terminal the extracellular region,transmembrane domain and the C-terminal intracellular region and the whole hydrophobicity is strong.The signal peptide analysis showed OPN5 is not secreted protein due to the lack of signal peptide.All these characteristics are consistent with the properties of the OPN5 as membrane protein.Sequence analysis showed that OPN5 consists of 7 transmembrane topology,one visual pigment(opsin)retinal binding site is underlined,one G-protein coupled receptors family and there is a Lysine residues on the 7th trans-membrane structure of the OPN5 which is sensitive to ultraviolet light.The analysis of phylogenetic relationship showed C.barabensis has a close relationship with C.griseus,Mesocricetus auratus and Mus musculus,while it is far from the primate and artiodactyla.These results are consistent with the traditional classification of species,which indicates the conservation of OPN5.Secondly,the relative expression of OPN5 mRNA and protein in brains of C.barabensis in different developmental stages was detected using real-time quantitative RT-PCR and immunohistochemistry.Additionally,FSH and LH levels in the blood were estimated using an enzyme-linked immunosorbent assay.The results show that:(1)At the m RNA level,the OPN5 expression in the brain was the highest in adult and significantly higher than that in the young and the old(P<0.05)of the male C.barabensis.The expression of OPN5 in the brain of female C.barabensis was highest both in subadult and adult,and was significantly higher than that of young and old individuals.At the protein level,the OPN5 expression in the brain was the highest in adult and significantly higher than that in the young and the old(P<0.05)of the male C.barabensis.The OPN5 expression in the brain of the female C.barabensis was highest both in subadult and adult,and was significantly higher than that in the old.(2)At the mRNA level,significant gender difference(P<0.01)was observed on the subadult individuals while there was no significant gender difference(P>0.05)in the young,adult and old.At the protein level,significant gender difference(P<0.05)was observed only in adult C.barabensis.(3)Hormone concentrations showed that the concentration of FSH in the blood of the male C.barabensis was the highest in subadult,which was significantly higher than that of adult and old(P<0.05)while the concentration of LH in blood was the highest in the young individuals which was significantly higher than that in the subadult,adult and old(P<0.05).For the female C.barabensis,the concentration of FSH in the blood is the highest in the young and subadult,which is significantly higher than that of adult and old(P<0.05).The concentration of LH in the blood of the young is significantly higher than that of adult and old(P<0.05).All results suggest that the expression of OPN5 mRNA and OPN5 protein and the concentration of FSH and LH in the blood have sex and developmental stage specificity.Lastly,the effects of photoperiod treatment on body weight,gonadal development,hormone concentration in the blood and the relative expression of OPN5 mRNA and protein,entration in the blood of the C.barabensis was investigated.The results show that:(1)At the end of one month photoperiod experiment,the C.barabensis exposed to artificial long day(LD)photoperiods had significantly higher(P<0.01;P<0.05)mean final body weights than the individuals in short day(SD)photoperiods for both males and females.On the whole,the weight of LD group was rising while the weight of SD controls was decreasing both male and female C.barabensis.(2)GSI was significantly higher both males and females in LD groups than that of SD groups.In males,significant difference(P<0.05)was observed in the weight of testes while no significant variations were observed in the weight of epididymis between the LD and SD individuals.In females,significant difference(P<0.05)was only detectable in the weight of ovary between the LD and SD groups and no significant was observed in the weight of uterus.(3)At the mRNA level,OPN5 mRNA expression in the brains increased significantly in the SD treatment group compared to LD controls(P<0.001)on the male and female C.barabensis.And significant gender difference(P<0.001)was observed on the LD groups while there was no significant gender difference(P>0.05)in the SD groups.(4)At the protein level,OPN5 protein expression in the brains increased significantly in the SD-treated group compared to LD controls(P<0.001)on the male and female C.barabensis.However,no significant gender difference(P>0.05)was observed both LD and SD individuals.(5)Significant differences(P<0.01,P<0.001)were observed in the FSH and LH concentrations of the blood between the male and female C.barabensis treated with different photoperiods.SD individuals had the lowest FSH and LH levels,while the LD group had the highest levels.All results suggest that photoperiod may be a direct regulatory factor of OPN5 expression,and the effect of photoperiod on OPN5 expression is different in sex.In addition,the photoperiod can affect the reproduction by affecting the hormone level,body weight and gonadal development of the C.barabensis.In summary,this study analyzed the effects of OPN5 on the reproduction of C.barabensis and the effect of photoperiod on hormone levels and body weight.This study plays a very important role in exploring the role of OPN5 in animal reproduction and has important theoretical significance. |
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