| Senescence is an unavoidable physiological process that is caused by aging of tissue and cells, declining of organ functions。Specific mechanism of aging is not very clear。Over the years, anti-aging scholars from around the world have done a lot of work, but also，many of the theories have been put forward. such as:Oxygen free radical theory, cell aging theory, Neuroendocrine-immune theory Etc. in all the theories, Oxygen free radical theory has been affirmed and recognized by most scholars. Currently, it has been widely used in brain aging, cataracts, reproductive aging and other animal models。With the advent of World Population Ageing, To Explore the mechanism of human aging, delay the occurrence of aging and improve the quality of life of older persons, has become an important scientific topics of widespread concern. We are also applying this model to produce sub-acute aging model in mice to observe the testis and sperm changes of aging mouse and to explore and perfect the mechanism of reproductive aging, to research the anti-reproductive aging mechanism of Resveratrol (RES), so as to provide more experimental evidences of Resveratrol anti-aging research。I purpose: To observe of the reproduction aging changes of the sub-acute D-galactose (D-gal) induced mouse and Resveratrol treatment。Ⅱmethods:36 C57/6Jmice of 3-4 months and body weight (25±2g) have been selected and randomly divided into six groups:normal Saline group (NS), aging model group (D-gal), Solvent control group(DMSO), High-dose Resveratrol group(RES-H), low-dose Resveratrol group(RES-L), And the positive control group(VC)。Aging model and drug treatment:①NS:Mice were given subcutaneous injection of normal saline for 8 weeks, after 2 weeks, they were given 6 weeks normal saline lavage;②D-gal:Mice were given subcutaneous injection of D-gal (120mg/kg) for 8 weeks, after 2 weeks, they were given 6 weeks normal saline lavage;③DMSO:Mice were given subcutaneous injection of D-gal (120mg/kg) for 8 weeks; after 2 weeks, they were given 6 weeks DMSO lavage;④RES-H:Mice were given subcutaneous injection of D-gal (120mg/kg) for 8 weeks; after 2 weeks, they were given 6 weeks RES-H(45mg/kg) lavage;⑤RES-L:Mice were given subcutaneous injection of D-gal (120mg/kg) for 8 weeks; after 2 weeks, they were given 6 weeks RES-L(15mg/kg) lavage;⑥VC:Mice were given subcutaneous injection of D-gal (120mg/kg) for 8 weeks; after 2 weeks, they were given 6 weeks VC(150mg/kg) lavage. And then observe the mice body weight, coat color, and the general situation.Supernatant were taken after Blood was obtained by the eye, And then were detected:①the serum superoxide dismutase (SOD) activity and the amount of MDA were Respectively detected by Xanthine oxidase method and TBA method。②The testosterone level in serum was detected by ELISA。HE staining, immunohistochemisty staining (IHC) of the mice testis paraffin sections:Part of the testis should be fixed well by 4% paraformaldehyde, and then use HE staining to observe morphological changes of seminiferous tubules after general dehydration transparency, wax dip, encasement and slice。the expression of P53 in spermatogenic cells were detected by IHC (SABC)。The determination of sperm quality:a part of epididymis sperm in mice were carefully made into sperm suspension and sperm smear, and then respectively measured as following:sperm motility rate with Eosin-staining; Sperm motility observed under light microscope; count sperm and calculate the density; However, sperm morphology in HE staining (deformity)。Electron microscope observation of Mice testis and sperm:The other part of mice sperm and testis tissue were fixed with 2.5%glutaraldehyde and then they were sent to electron microscope room:ultrathin sections of testicular tissue and sperm were respectively observed with transmission electron microscope and Scan electron microscope (SEM)。III Results:(1) body weight, coat color, and general changes in mice:Comparing with NS group, the mice in D-gal group and DMSO group have a characteristic of slow weight increment, lusterless coat color,even yellow or white color, some hair loss conditions in mice also appeared。They also have a worse General situation:lethargy, reduced activity, eating drinking; mice in RES-H group and VC group have a similar performance with NS group:the aging signs have been improved to a certain extent, Coat color and the general situation is better; the RES-L group mice have a body weight, coat color, and general changes in between the D-gal group and the RES-H group。(2) changes of serum testosterone (T) level, SOD activity and MDA amount:Serum SOD activity & T level:comparing with D-gal group, there are no significant difference between the D-gal group and RES-L group, but D-gal group have an significantly lower T level than the NS group (P<0.05); The RES-H significantly increase mice’s serum testosterone levels (RES-H group VS the D-gal group, P<0.05)。serum MDA amount:D-gal group was significantly higher than the NS group and the RES-H group, while the VC group and the RES-L has no significant difference (P> 0.05)。(3) HE staining of mice testis paraffin section:The seminiferous tubule in D-gal group appeared obviously morphological changes under light microscope:Seminiferous tubule basement membrane thickening part, incomplete, Seminiferous tubule germ cells decreased, arranged in sparse; comparing with the D-gal group, the RES-L group mice have no significant improvement while the RES-H group and VC group mice’s Seminiferous tubule improved obviously:Almost the compete basement membrane, increased germ cell, however, comparing with the NS group, Spermatogenic cells remained relatively sparse。(4) P53 immunohistochemistry of testis paraffin section。p53 positive cells were mainly Spermatogenous cells positive products localized in the nucleus。D-gal group was significantly higher than that of p53-positive cells rate in NS group(P< 0.01); RES-H group were P53 positive cells rate was lower than D-gal group (P<0.05); while,The VC group and the RES-L was no significant difference each other (P> 0.05)。Group P53 D-gal positive cell rate was significantly higher than the NS group (P<0.01), RES-H group were P53 positive rate of less than D-gal group (P<0.05)。(5) changes in sperm quality in mice:As for sperm quality, D-gal mice was significantly lower than the NS group (P <0.01), it shows that aging can cause the overall decline in sperm quality, including lower sperm density, motility, viability, increased malformation rate, etc. D-gal mice was significantly lower than the NS group of sperm quality (P<0.01), This shows that aging can cause the overall decline in sperm quality in mice; VC group were much higher than the sperm abnormality rate vs NS group and the RES-H group while of sperm vitality there were insignificant difference among the D-gal group, DMSO group, RES-L group and VC group that VC and RES-L can not be a good increase in the aging mouse sperm vitality.(6) changes in sperma cell apoptosis rate in mice:The mice in D-gal group has significant difference comparing to NS and RES-H group, it show that RES-H could induce sperma cell apoptosis rate; it is same to say:though the inducing sperma cell apoptosis, RES has effect on anti-reproduction aging。ⅣDiscussion:During the experiment, we successfully established a D-galactose induced aging mice model, we also observed mice’s Body weight, coat color and general situation, and reproductive organs changes。We draw several conclusions。aging signs of model mice:mice showed that Yellow coat color, worse general situation, Increasing free radicals, lipid peroxides accumulation and decreased antioxidant capacity; Mice testis and sperms have morphological and ultra-structure change; serum T level deceased; seminiferous tubules Structure disordered; Spermatogenic cell were of decreased proliferation and increased apoptosis; high expression of P53 gene so that testis spermatogenesis and reproductive function of aging mice decreased.Resveratrol can increase serum SOD activity, T levels, P53 gene expression and reduce the amount of serum MDA and spermatogenic cell apoptosis,so as to Improve the body’s antioxidant capacity and reproduction function。Our conclusion were as following:resveratrol has a good anti-aging effects, not only it can Improve the aging body’ antioxidant capacity and testis Endocrine function, but also Promote spermatogenesis function and decline of reproduction function。Summary:resveratrol has an distinct effect on aging reproduction function in male mice。... |
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