| Meiosis is the important process of male’s spermospore differentiation to sperm, which normally occur andlargely dependent on homologous chromosome synapsis, exchange and separation of meiotic prophase I, and is closely related to genetic recombination. Studies showed that the abnormal of MI will make the meiosis disorder, stagnation, and even lead to azoospermia. Therefore, it will contribute to the understanding of mechanism of spermatogenesis of meiotic recombination frequency and associationanalysis, so as to reveal the causes of male infertility.With the rapid development of the technology on cancer treatment, the survival rate of male patients with cancer is rising and their demand for fertility preservation is increasing. Therefore, it is of concern how to preserve the fertility of male cancer patients safety and help them get healthy offspring, as well as common problem currently facing on reproductive medicine and oncology.In order to achieve the ultimate goal of prolonging life and fertility preservation patients balanced,clinicians must consider the toxicity and safety of male germ cellantitumor drugs first when they in the formulation of anti cancer regimens.Among anti-tumor drugs, there are two representative drugs: traditional antitumor drug trichostatin A(Trichostation A, TSA) and a new anti cancer drug GSK2126458. At present, these two kinds of drugs have been used in clinical trials of tumor treatment, but the effect on spermatogenesis have not been reported at home and abroad?The cryopreservation of testicular tissue has become another important means of male fertility preservation.But there still was many problems on freezing testicular tissue itself, such as the methods of frozen testicular tissue(with programmed slow freezing and rapid freezing).There is no unified standard, there is controversia on the choice of cryoprotectant. About the effects of different cryopreservation methods on the process of meiosis of human testis spermatocyte, there are only a few reports about the program of slow freezing, but there is no report on rapid freezing. Therefore, the study on the influence of spermatocyte genetic recombination of antitumor drugs and rapid freezing not only can evaluate its safety, but it is also the basis for male patients to choose the best time for fertility preservation and cryopreservation methods provide. Objective:We study the effects of different concentrations of anti tumor drugs TSA and GSK2126458 on male mice’ spermatogenic function,spermatocyte meiotic recombination sites and synapsis fidelity with immunofluorescence; and the differential expression of MLH1 gene of spermatocyte meiosis in testis before genetic recombinationand later split from different human testes before the testis frozen and after. Designed to assess the safety of the two kinds of anti tumor drugs and rapid freezing method, and provide for clinical application. Methods:1. The effect of anti tumor drugs TSA and GSK2126458 on the spermatogenic function of male mice. 18 mice were randomly divided into group TSA(0.1)(3rats); TSA(0.2)(3rats); GSK(0.1)(3rats);GSK(0.2)(3 rats);control group(6rats). Analyzed homologous chromosome genetic recombination and synapsis fidelity with one part of each testicular tissue by spreading and immunofluorescence; and the other part of the tissue were slice prepared and HE stained. We observed the sperm density and survival rate with the epididymal fluid of each group.2. The effects of rapid freezing on the process of meiotic genetic recombination of the spermatocyte of human.The testis from four patients with prostate cancer were divided into two parts respectively.One part were cooled,then they were both analised homologous chromosome synapsis and genetic recombination fidelity by spreading and immunofluorescence3. The expression of MLH1 gene from three kinds of testis. Divided into obstructive azoospermia group(n=8), non obstructive azoospermia group(n=14) and normal spermatogenesis group(n=12). We analyzed the expression of MLH1 gene in each group with RT-PCR. Results:1. The number of MLH1 on each cell of the group of TSA(0.1) was significant higher than the control(P<0.05), and there were no significant statistically difference compared with TSA(0.2)(P>0.05). The number of SC which has zero MLH1 locus of the two TSA group were lower than the control group(P<0.05, P<0.05),and the average number of SC which has one MLH1 locus were higher than the control group(P<0.05, P<0.05). The weight of left testis in the mice of the group of TSA(0.1) was lower than the control group significantly(P<0.05).2. There was no significant difference on the number of MLH1, SC which has zero to three MLH1 locus, the ratio of cells with MLH1 loci splits in chromosome XY, gaps, mice body weight, testis weight, sperm density,sperm motility and sperm motility between the GSK groups(P >0.05).3. The number of MLH1 in eyery cell of the two TSA groups was significantly higher than that of two GSK groups(P<0.05, P<0.05). The number of SC which has zero MLH1 locus were lower than the other three groups significantly(P<0.05), while the number of SC which has one MLH1 locus was higher than the other three groups significantly(P<0.05).4. The recombinant frequency of meiosis and synapsis fidelity have no significant difference in same testicular of fresh and frozen thawed(P >0.05).5. The expression of MLH1 in the two azoospermia groups are higher than that of normal spermatogenesis significantly(P<0.05); and the expression of MLH1 in the group of non obstructive azoospermia was higher than that of obstructive azoospermia group significantly(P <0.05). Conclusions:1. The recombination frequency in the process of spermatocyte meiotic of male mouse were increased in two TSA groups.2. There were no effects on the recombination frequency and spermatogenic function in the process of spermatocyte meiotic with the two groups of GSK2126458.3. The effects of TSA on the recombination frequency of male mouse is greater than GSK2126458.It is suggested that clinical antitumor drug treatment men Prefer GSK2126458.4. There were no effect on the recombination frequency and spermatogenic function in the process of spermatocyte meiotic with quick frzeeing,and it would be the effective method for male fertility preservetion.5.The gene named MLH1 in the human testicular of azoospermia is over expression, and may mediate during the Process of spermato genesis. |
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