| Background and objectiveMale infertility is a social and health problem in worldwide, with complexcauses. It’s clear that spermatogenesis obstacle leaded by selenium deficiency andexcess is one of its causes. Selenium is an important trace element for animals,playing a biological role mainly through its selenoprotein forms. Until now, a lot ofstudies have revealed damage to the reproductive system structure and function byselenium deficiency or excess, but the mechanism has not been clarified clearly. It’seven rarely reported on the expression of selenoproteomes regulated by seleniumlevels in male reproductive system, selection of specific-function seleniumprotein,and futher study of the effects of selenium on male reproductive health.Studies have indicated that semen selenium levels are generally low in infertilemen, and the higher semen selenium levels, the more sperm and the stronger motilityin the appropriate selenium concentration range. Containing large amounts ofunsaturated fatty acids, human sperm cells are vulnerable to reactive oxygen species(Reactive oxygen species, ROS) in semen, and thus induce lipid peroxidation,generate malondialdehyde (Malonaldehyd, MDA) and other substances, which candamage sperm membrane, decrease sperm motility, week its function, and finallyresult in infertility. Selenoproteins represented by Glutathione peroxidases(Glutathione peroxidases, Gpxs) are powerful in antioxidant for eliminating excessfree radicals and possibly inhibiting lipid per-oxidation, and thus play an importantrole in protecting sperms from oxidative damage and improving sperm motility. Up to now,25Selenoproteins in human and24in rodents are found. Most of thediscovered functions are glutathione peroxidase family (Gpxs,5species), deiodinasefamily (Iodothyronine deiodinases, Dios,3species), thioredoxin reductase family(Thioredoxin reductases, Txrnds,3species), selenium phosphate synthase2(Sps2),and selenoprotein P (Sepp1). Functions rarely reported and not fully confirmed,include selenoprotein15-kDa (Sep15), selenoprotein H (Selh), I（Seli）, M（Selm）,N（Sepn1）, R（Selr）, S（Sels）, T（Selt）, W（Sepw1）,while function of selenoproteinK（Selk）, O（Selo）, V（Selv）are completely unclear. There is a popular consensusthat selenium acting as oxidoreductase, selenoproteins enable selenium to carry out avariety of biological functions. They also play an important and even a key role inenhancing immunity, anti-aging, preventing cardiovascular disease, cancer, andmaintaining man reproductive function mainly through their own activity.The testicle is the vital organ for secreting male hormones (particularlytestosterone) and germ cell generation, including seminiferous tubules and Leydig.The seminiferous tubules are mainly formed by spermatogenic cells and supportingcells. Spermatogenic cells are the general name for spermatogonia, primaryspermatocytes, secondary spermatocytes, spermatids and sperm.It is a process calledspermatogenesis that these cells move from the basement membrane towards thelumen, get mature and move into the lumen. It’s clearly that testicle is an importanttarget organ of Se, for example, essential phospholipid hydroperoxide glutathioneperoxidase (Gpx4) acts as an antioxidant enzyme in the early period ofspermatogenesis, and transforms into essential structural protein component withoutactivity in mature sperm. Sepp1provides most Se for testicle through the ApoER2receptors in epithelium of the testis. Some studies also suggest that Seps1, Sep15,Txnrd3and other selenoproteins play an important role in maintaining malereproductive function. In2010, a paper published by British scientists shows that Selv(human gene symbol SELV, rodent gene symbol Selv) is specifically distributed intesticle and mainly in the late stage of spermatogenic cells. It also suggests that Selvhas some important functions in the process of spermatogenesis, but the knowledge ofits functions is still blank. As mentioned earlier, Se is a structural component of selenoproteins, but not alltissues and selenoproteins will be affected by selenium defeciency. Someselenoprotein mRNA shows nonsense-mediated degradation (Nonsense-mediateddecay, NMD) in selenium deficiency, and thus causes significantly reduction ofselenoprotein mRNA, but there are three hierarchical rules preventing specificselenoproteins from being affected:(1) selenoproteins in the brain and endocrinetissue enjoy priority in utlizing Se;(2) the expression of important selenoproteins ispreferentially sustained;(3) selenoproteins important to reproductive system and sexget preferential expression. The potential mechanism regulating the expression ofthese selenoproteins is a hot molecular biology-based research. Testicular Gpx4simultaneously satisfy these three classic level rules, and dietary Se hardly affects itsexpression. It is possibly because Sepp1in the blood-testis barrier bufferes Seconcentration fluctuation, or Gpx4located in testicular cells has special mechanismsfor avoiding influence caused by Se fluctuation. Selv is specifically distributed inspermatogenic cells, but its functions are yet to be revealed. It needs research frommRNA and protein to confirm whether testicle can keep Selv expression from beingaffected by Se fluctuation.At present, we has achieved significant results in using selenium preparation toprevent people from selenium deficiency disease, but the frequent occurrence ofselenosis is due to blindly excess of selenium in the treatment. The project will carryout animal experiments to discover expression patterns and potential function ofselenoprotiens in rat testicle at different trophic levels of body selenium, to explorefunctions of Se and selenoproteins in spermatogenesis and male reproductive health,and provide theoretical basis in prevention and treatment of Se and selenoproteins formale infertility.Methods1. Experimental methods1.1Design of experimental groupsThe30four-week-old male rats were fed selenium deficiency diet for five weeks to establish selenium deficient animal model. Then they were randomly divided intofive groups of each six rats: selenium deficeincy model group (BD5w group),selenium deficiency control group (BD9w group), selenium adequate group (BD+0.25ppm Se group, denoted BD+0.25group), high selenium group (BD+3.0ppm Se group,denoted by BD+3.0group), selenium poisoning group (BD+5.0ppm Se group,denoted by BD+5.0group). BD5w group were killed at age of9weeks. The otherfour groups were continued to be fed corresponding selenium diet for another fourweeks.1.2Measurement of animal growth performanceWeekly recorded the animal weight; when animals were sampled, weighedtesticular and calculated testicular index.1.3Determination of selenium concentration in tissues, blood cells of ratCollected Heart anticoagulated whole bloodand blood cells after centrifuging;isolated testis, liver, kidney from rat body. After all samples were digested andprocessed, detected selenium concentrations with inductively coupled plasma massspectrometry (ICP-MS).1.4Determination of plasma8-hydroxylation of deoxyguanosine of ratAccording to the kit instruction, measured8-hydroxylation of deoxyguanosine(8-OHdG) concentration of rat plasma.1.5Observation of sperm quality and ultrastructure of ratKilled rats of selenium deficiency control group, BD+0.25group, BD+3.0group,BD+5.0group, and then separated epididymal to collect sperms with capacitationregents. Observed sperm under the microscope (×400) for its density and spermmotility, and made video recording; drew a drop of12%neutral formalin-fixedspecimens on a glass slide to prepare sperm splices and observed sperm morphology(×400); centrifuged sperm suspension to obtain sperm sediment, then fixed with2.5%glutaraldehyde, prepared the conventional electron microscopy splices, and observedthe ultrastructure of sperm under the transmission electron microscope.1.6Determination of selenoproteomes expression in rat testis (RT-qPCR)Homogenized small testicular tissue by micro-homogenizer, used RNAiso Plus reagent to extract testis total RNA, and compounded first-strand cDNA, a template forqPCR, to assay mRNA level of the testis selenoprotein genome.2. Statistical methodsSAS9.0software was used for statistical analysis. ANOVA (One Way Anova)was used for groups compare, and Bonferroni method for further comparison betweentwo groups. Statistical differences of p-values at the level of0.05or less wereconsidered significant.Results1. Effects of selenium on testis development in rat1.1Effects of selenium on weight and testis index of ratThe selenium deficiency and excess (3.0and5.0ppm Se) diet could lead to slowgrowth and loss of weight, weight gain speed descended as: BD+0.25group>BD+3.0group>BD+5.0group> selenium deficiency control group; testis weight and index ofBD+5.0group were slightly higher than the rest of the three groups, but the differencewas not statistically significant (P>0.05); selenium deficiency control group, BD+0.25group, and BD+3.0group were not significantly different (P>0.05).1.2Effects of selenium on selenium concentration in rat tissuesRats fed on selenium deficiency diet could cause significant decline of seleniumconcentrations in the testicle, liver, kidney, blood cells; these were lower than those ofselenium adequate group (P<0.05) when Rats fed selenium deficiency diet for5and9weeks; testicular selenium concentration is significantly higher than the other tissuesunder selenium deficiency status(P<0.05); tissue selenium concentration increasedafter supplementation of different selenium doses; selenium concentration of liver,kidney were positively correlated with increasing doses of selenium, while testisselenium concentration increased slowly with increasing doses of selenium especiallywhen the selenium dose was beyond3.0ppm.1.3Effects of selenium on8-OHdG in plasma of ratThe8-OHdG levels among groups descended as: selenium deficiency controlgroup> BD+5.0group> BD+3.0group> BD+0.25group, and the difference between BD+0.25and BD+3.0group was statistically significant (P<0.05), the differenceamong BD+0.25group, BD+3.0group, and BD+5.0group was insignificant(P>0.05).2. Effects of selenium on sperm quality and ultrastructure in rat2.1Sperm qualitySperm density: selenium intervention groups descended as: BD+0.25group>BD+3.0group> BD+5.0group> selenium deficiency control group; BD+0.25groupis1.75times of the selenium deficiency control group (P <0.05); BD+3.0group is1.57times of the selenium deficiency control group (P<0.05).Sperm motility: for progressive motility(PR), BD+0.25selenium group wassignificantly higher than the selenium deficiency control group (P<0.05), BD+3.0group and BD+5.0selenium group were also higher than the selenium deficiencycontrol group, but the difference was not statistically significant (P>0.05); fornon-progressive motility (NP), selenium deficiency control group was significantlyhigher than BD+0.25group, BD+3.0group, and BD+5.0group (P<0.05); for totalmotility (PR+NP), there was no significant difference among the selenium deficiencycontrol group and the other three selenium intervention groups (P>0.05).Sperm morphology: sperm abnormality rate of the BD+5.0group was the highestand the next is selenium deficiency control group; BD+0.25group and BD+3.0groups were similar. To compare BD+5.0group with BD+0.25group and BD+3.0group, the differences was statistically significant (P<0.05); compared with theselenium deficiency control group, the difference was not statistically significant(P>0.05).2.2Spermatozoa ultrastructureSelenium deficiency control group (BD9w group) appeared nucleus gap innuclear membrane and no acrosome; membrane folds appeared in the middle part ofsperm. In some areas, mitochondrial was disorder and void; some mitochondriabecame obvious empty. In selenium adequate group (BD+0.25group), sperm headhad nuclear membrane integrity; tail section had smooth and intact membrane; mitochondria arranged in concentric circles with compact structure and most werenormal in shape and size; the number of vacuolar mitochondria decreased; axis andperipheral dense fibrous were in alignment. In high selenium group (BD+3.0group),the sperm nuclear membrane was intact and the sperm acrosome was not obvious;many huge mitochondria was in the middle part of sperm; some mitochondriasbecame vacuolar; outermost membrane disappeared; some sperm mitochondriaappeared loose and ill-defined in the peripheral portion of the tail section. In seleniumpoisoning group (BD+5.0group), sperm head membrane wrinkled, and acrosomewasnot obvious; peripheral nine dense fibrous was not intact in structure; membrane wasnot clear in the middle part of sperm; most mitochondria became vacuolar; somefibrous sheath section in sperm tail became loose; mitochondria lost and spermmembrane was not clear. Overall, sperm ultrastructure in selenium deficiency controlgroup and selenium poisoning group got the greatest harm, followed by high-seleniumgroup. Although adequate selenium supplement group could restore spermultrastructural damage caused by selenium deficiency in a certain degree, there wasstill some part of the damage failing to be restored.3. Effects of selenium on expression of selenoproteome in rat testisThis test used RT-qPCR method to quantify testicular selenoproteome mRNAlevel. The results revealed that testicular selenoproteome was significantly affectedafter selenium intervention; selenoproteome generally declined in the seleniumdeficiency groups;10gene mRNA abundance in different supplementary seleniumdoses were found significantly up-regulated (P<0.05) and statistically significant. Thegenes achieving the highest expression included6genes in BD+0.25group (Gpx1,SnGpx4, Txnrd2, Dio1, Seli, Sepn1),3genes in the BD+3.0group (Selr, Sepp1,Sepw1), and1genes BD+5.0group (Txnrd1).6genes were statistically significantand more than2times the expressed difference among groups (Gpx1, SnGpx4, Txnrd1,Txnrd2, Seli, Selr). Txnrd1, SnGpx4, Selr are most sensitive to selenium regulation.Conclusion1. Selenium of deficiency or excess（3.0and5.0ppm Se）can cause developmental retardation, damage of DNA oxidative, disorders of sperm production, spermstructural damage, increase of deformity rate, decrease of sperm progress motility, anddecrease of the expression of multiple selenoprotein in testis.2. Selenium supplement (0.25ppm Se) can effectively improve the development ofrats, reduce DNA oxidative damage, recover sperm structural damage, improve spermquality, and up-regulate selenoproteomes mRNA level.3. When selenium is deficient or excess in rat, abnormal expression of testicularselenoproteins leads to decrease of sperm quality and structure damage. |
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