Empirical Study And Mechanism Exploration Of Dutasteride Treatment On Rat Epididymal Sperm Maturation And Fertility

Aim:To study the effect of dutasteride on rat epididymal sperm maturation and fertility andsearch for the post-testicular targets associated with regulation of male fertility,simultaneously, to establish the epididymal gene expression profiling database aboutfertility regulation, approaching the possible mechanism of action on male antifertilityby dutasteride.Methods:1. Male rats were treated with 20 mg/kg/d and 40 mg/kg/d dutasteride, meanwhile,with 10mg/kg/d alpha-chlorhydrin as a reference group, for 14 days. Each male ratwas paired with 2 female rats in proestrus when administration terminated. Femalerats were examined the next morning for the presence of sperm in vaginal smears andunderwent a cesarean section on day 13 of gestation. Then the reproductive indiceswere calculated as follows: copulation index, pregnancy index, and fertility index,which can evaluate male fertility directly. The sperm motility and morphology wereevaluated by computer-assisted sperm analysis (CASA), moreover, the caudaepididymal sperm ultramicrostructure were analyzed by transmission electronmicroscope(TEM), which can evaluate the influence on sperm maturation bydutasteride ; sperm survival rate was assessed by SYBR-14 and propidium iodide (PI)fluorescent staining; serumal dihydrotestosterone (DHT) and testosterone(T) of ratswere detected by enzyme-labeled immunoassay (Elisa), which can evaluate the effecton androgenic levels; the weights of testes and epididymides, were determined byprecise electronic balance; histological examination of above tissues were evaluatedby HE staining and TEM, which can evaluate treatment on histology of reproductiveorgans by dutasteride.2. We carried out the rat epididymal epithelial primary cell culture, using the syntenicmethods of differential adherence separation, filtration of stainless steel strainer meshand addition of dihydrotestosterone; then determined the proliferation activity ofepididymal epithelial cells and drawn the cell growth curve; finally identified the purity of cultured cells by immunologic cell staining using antibodies of keratin andvimentin.3. We determined the in vitro effects on rat epididymal epithelial cell proliferationactivity by various dosage dutasteride using Cell counting kit-8(CCK-8). In addition,we also determined the dutasteride treatment on in vitro activity of sialic acid andα-1, 4 glycosidase secreted by epididymal cells. Finally, we evaluated the in vitroinfluence on epididymal epithelial cell morphology by TEM analysis.4. We studied the differential epididymal gene expression among the dutasteride,alpha chlorhydrin and control groups using Affymetix Genome Rat 230 2.0Oligo-microarray. First, we extracted 9(3 of each group) epididymal RNA samplesusing Trizol, determining the quantity and quality of RNA applying optical desity(OD)and denaturing gel electrophoresis, then the RNA samples were mixed and purified;next we synthetized and purified cDNA using Affymetrix one-cycle cDNA SynthesisKit and Affymetrix one-cycle cDNA Synthesis Kit; then cRNA was synthetized,biotin labeled and purified GeneChip IVT Labeling Kit and Genechip SampleCleanup Module; furthermore, qualified cRNA gragmentation process was done; nextstep was to carry out the probe hybridization in the Genechip hybridization oven 640,16 hours; after eluted and stained, arrays were put into GeneChip Scanner 3000 forscanning, using GeneChip operating software(GCOS) to analysis the presentation.Then the probe signals of 3 groups were normalized and compared, which screenedthe differential expressed genes between each group. We performed followingfunctional classification for differential genes. Finally, gene expression validationwere executed by Real-Time quantitative polymerase chain reaction (PCR)Results:1. The serumal DHT levels of dutasteride treated animals were markedly decreasedcompared with the control group (P＜0.01). Accordingly ,the motility of spermatozoafrom the cauda epididymidis of dutasteride treated rats showed a significant decreasein percentage of motile, progressively motile sperm and sperm survival rate(P＜0.01or 0.05), At the same time, the morphology of cauda epididymal spermatozoa wasalso adversely affected by the treatment. Finally, female rats mating with treatedmales resulted in fewer successful pregnancies and embryos. At the same time, theserumal T levels and copulation index were not apparently changed, the histology oftestis and epididymis were also not affected by HE staining. Nevertheless, we found there had nuclear chromatin aggregation and bulk of vacuolation in the epididymalepithelial cells from dutasteride group using TEM analysis, which shown the cell hadapoptosis premonition.2. The rat epididymal epithelial cell culture was performed for 13 times in total ,whichhad 9 times successful culture and a successful rate of 69.2%; then we carried outsubculture for 9 times, which had succeeded for 5 times and a successful rate of55.6%;finally the immunological cell staining result was that endochylema ofepididymal epithelial cell shown brown by keratin staining, on the contrary, cellstained by vimentin exhibited negative, which have the purity of over 95% and couldbe applied for in vitro functional study.3. The proliferation activity, morphology and function of rat epididymal epithelial cellcan be affected by dutasteride action. As to IC₅₀ for cell proliferation, it is 16.1μg/mlwhen the epithelial cell cocultured with dutasteride, supposing that dutasteride havean strong inhibitory activity on cell proliferation; moreover, dutasteride can inhibit thesialic activity; furthermore, duasteride is also able to induce cell apoptosis.4. Using Affymetix Rat Genome array 230 2.0, we studied the differential ratepididymal gene expression between dutasteride high dose group and control group.According to the "Present" by detection p-value＜0.04, there is 17031 transcriptsdetected among the 31042 genomic probe sets in control group, in proportion to54.8% for total transcripts; meanwhile 18251 transcripts detected in dutasteride groupand have a proportion of 58.7%. Then we screened the differential expressed genesbetween two groups using GCOS, GeneSpring and Fatigo+. Compared to controlgroup, there is 950 down-regulated genes and 1996 up-regulated genes in dutasteridegroup, which are involved in macromolecular metabolism and transport, cellcommunication, defense reaction, receptor activity, ion combination, cell apoptosis,hydratase and transferase activity. At the same time, we also studied the differentialrat epididymal gene expression between chlorhydrin group and control group. Thereis 17410 transcripts detected among the 31042 genomic probe sets in chlorhydringroup, in proportion to 56.0 for total transcripts. Compared to control group, there is79 down-regulated genes and 90 up-regulated genes in chlorhydrin group, which areinvolved in macromolecular metabolism and transport, primary metabolism process,cell metabolism, biological process regulation, immunology regulation, ioncombination, hydratase and oxidoreductase activity. It's very important that we find many genes relation to the cell apoptosis, which is consistent with our former study.Finally, the gene expression level validated by RT-PCR is coherent with thehybridization analysis results.Conclusions:1. There have a correlation between epididymal sperm motility and the inhitory forserumal DHT level, more inhibiting making lower sperm motility and fertility. At thesame time, dutasteride has less influence on testicular morphology and function. Sowe suppose that DHT inhibiting by dutasteride have an effect on epididymal spermmaturation, then prohibiting the sperm-egg fusion and achieving contraception. Inaddition, we also found that dutasteride can induce epididymal epithelial cellapoptosis, which also can affect cell secretary activity and change the epididymalmicroenvironment, leading to dysfunction of sperm maturation and fertility. This issimilar to it's inhibitory effect on prostatic cell proliferation and treatment on BPH. Toour knowledge, it is the first report that dutasteride cause rat infertility at specific doseand durations. This animal sterility model can be used for studying androgen-regulated of epididymal function (including screening fertility-related genes) andinvestigating neotype male contraceptives.2. As to the primary rat epididymal cell culture, the achievement ratio is relatively lowbecause of complexity of operation process, interruption of interstitial cell,uncertainty of adherent separation and limitation of cell subculture, which increasesthe difficulty of pharmacological research in vitro. In present circumstance, wesuppose there be more trying to develop the cell cultural system.3. The results suppose that dutasteride has a strong inhibitory action on cellproliferation in vitro. In addition, dutasteride maybe affect epididymal secretedactivity through inhibiting the sialic acid. Finally, it is more important that dutasteridecan induce cell proliferation and interrupt it's function, which can furthermoreinfluence epididymal microenviroment and sperm maturation, inhibiting sperm-eggfusion and male fertility.4. We studied the differential gene expression between drug group and control group,screening some down or up regulated genes. After performing gene functionalclassification, we primarily establishe the epididymal gene expression profilingdatabase associated with fertility regulation and may find out new epididymal fertilitytargets in order to provide practical an theoretic bases for development of new male contraceptives.