The Influence Of FMR1Gene Knockout On The Male Mouse Reproduction

In last half century,with the development of society, the incidence of infertilecouples increased year by year. In2004, statistics from European ReproductiveAssociation showed that: accounted for25%of couples of childbearing age can notbecome pregnant within one year, accounts for50%of infertility due to male factors.According to WHO, the male infertility factors include varicocele(12.3%), sexualdysfunction(1.7%), reproductive tract infections(6%), congenital abnormalities(2.1%),acquired diseases(2.6%), endocrine disorder(0.6%), immune factors(3.1%) and otherdiseases(3%). But pathogenesis in more than70%of male infertility is still unknown,which makes the diagnosis and treatment of male infertility complex. In recent years,some new discovers about the genetic cause of male infertility have been proposed.Genetic defects also may lead to male infertility, such as X-chromosome genetic, Ychromosome microdeletions in the azoospermia factor district, autosomal abnormalitiesor other known gene mutations. However, only15%cases can be explained by the abovefactors. So far, study of male infertility is becoming very important.In patients with primary infertility, spermatogenesis disorder is the main type. Sothose genes related closely to spermatogenesis are research hotspots. Humanspermatogenesis is a extremely complex process that involves proliferation of primordialgerm cells, meiosis, cell differentiation, etc. It is estimated that more than4000genes are involved in the process of spermatogenesis. In recent years, many genes closely relatedto spermatogenesis are identified, but whether those genes are associated with maleinfertility requires further experimental study.Fragile X syndrome(FXS) is one of the most common inherited mental retardationdisease, the incidence is second only to Down syndrome, accounting for2%-6%ofnon-specific mental retardation,40%of the X-linked mental retardation. FXS patientshave varying degrees of mental retardation, attention disorders, easy to epilepsy seizures,personality changes, hyperactivity, autistic and other abnormal social behavior, a specialface and giant testes(one side is more than25ml) after puberty, low male fertility andpremature ovarian failure in adult females. Fragile X syndrome results from atrinucleotide repeat (CGG)n expansion in the5-untranslated region of the fragile Xmental retardation gene1(FMR1) and subsequent hypermethylation which lead totranscriptional silencing of FMR1and loss of its encoded protein FMRP (fragile Xmental retardation protein).In order to find the complex mechanism of FXS, and givebetter treatment to FXS patients, reproductive systems and mental behavior-related issuesare being studied.In the study of FXS, the FMR1gene knockout mice model is widely recongnized asthe best research model of FXS, a variety of performance are quite similar to theabnormalities of patients with FXS, such as excessive activities, learning disabilities,testicle increases after puberty, so we try to learn the influence of FMR1on malereproduction through studying the reproductive system and function of FMR1geneknockout mouse.PurposeTo investigate the influence of fragile x mental retardation-1gene on thereproduction of male mice. And put a foundation on further study of the influence ofFMR1gene on sexual function and reproduction system, spermatogenesis regulation andpossible treatments.Materials and Methods1.Experimental animals: FMR1gene knockout(KO) and its wild type(WT) FVB inbred strain mice, after genotype identification by PCR to detect the FMR1genesegments, will be fed and bred by Experimental Animal Center of Guangzhou MedicalUniversity.2. Animal grouping:2.1Reproductive experiments and methods: in total of72mice.12FMR1knockout(KO)male mice and12wild type(WT)male mice will be separately put in one cagewith2wt female mice(in total of48mice) for5days for mating,observe the sexualbehavior of male mice, record the first time to capture the female after they are put in onecage (latent period of capture), frequence of male capture female within30min, thefemale pregnancy rate, number of litters are counted,and male mice having offspringscalculated. Collect the testes and epididymides of12mice in each group, weigh andcalculate the organ coefficient. The density, motility and morphology of one caudaepididymis sperms are analysed. Serum T, FSH and LH concentrations are alsomeasured.2.2Histology and methods: in total of10mice. Collect the right testis andepididymides of5adult mice in each group, perform with HE staining. Observe thehistology with a high power optical microscope.3.Statistical method: All the data results are presented in form of, comparingthe KO and WT group with independently sample t-test by software SPSS15.0. There arestatistical significance if P<0.05.Results1.Result of testes and epididymides organ coefficient comparison: The KO testesand epididymides weight are significantly higher than the WT group. No significantdifference of the body weight between KO mice and WT mice. KO mice testes andepididymides coefficient are significantly higher than those of WT mice.2.Reproductive experimental results: The capture latent time of KO mice weresignificantly prolonged compared with WT mice(15.76±6.14min vs.9.24±5.32min,P<0.05), the frequence of capture within30min of KO mice wassignificantly lower than WT mice. Male fertility showed that41.7%of KO mice had pups whereas in the WT group91.7%of the mice had pups(P<0.05), The pregnant rateof WT female mice mated with KO males was significantly lower than the controlgroup(41.7%vs.87.5%, P<0.05), the average litter size of KO and WT respectivelywere6.50±2.27and8.22±3.03(P>0.05), no significant difference.3. Sperm analysis: two groups of mice in sperm count, motility, and abnormalsperm ratio were not statistically different.4. Sexual hormones results: No significant difference were found in serum T, FSHand LH between two groups.5.Testes and epididymides histological observation: there were no significantdifference in seminiferous tubule diameter, structure, thickness of the seminiferoustubule epithelium between KO and WT mice. The morphology and number of sertolicells, spermatogonia, spermatocytes of KO group had no difference with WT.Epididymis morphology and its epithelial thickness of KO mice showed no obviousabnormality.ConclutionsWe can conclude that FMR1gene may play an important role in the development ofmale genital system. The absence of FMR1would increase the weight of testes andepididymides, reduce the reproduction of male mice. But no obvious abnormal wasfound in morphology of testes and epididymides. The spermatogenesis of FMR1micealso seemed not being affected. The exact mechanism through which FMR1geneinfluences the male genital system needs further studies.