The Effect Of ART On The Gene Expression Of Testis And Lipid Metabolism Of Male Mice And The Mechanism Involved

Part I The persistency and intergenerational transmission of differentially expressed genes in the testes of ART miceWorse reproductive health in the men born through intracytoplasmic sperm injection (ICSI) or other assisted reproductive techniques (ART) has been reported in some studies, such as hypospadias, reduced serum testosterone level, and lower semen quality. However, because of the interference of genetic and many environmental factors, it is difficult to identify whether ICSI methods affect male reproductive health based on clinical human data. Therefore, we established mouse ART models using inbred mice, including controlled ovarian hyperstimulation (COH), in vitro fertilization (IVF) and ICSI mice. Semen quality, serum testosterone and histological analysis of testes were done but no significant changes were shown in our previous studies. In this study, the gene expressions in testes were analyzed in ART mice.The gene expression profiles were analysed in the testis of both ICSI and naturally conceived (NC) newborn F1mice using micro-array analysis. Among the differentially expressed genes, we focused on the expression of8male (Dmcl, Prok2, Rec8, Rhox5, Spol1, Adcyaplr1, Amh and Pdgfra) reproduction-related non-imprinted genes and6paternally expressed imprinted genes (Igf2, Kcnqlot1, Plagl1, Snrpn, Peg3and Mest). Quantitative real time RT-PCR (qRT-PCR) was used to analyse the expression of these genes in mouse testes of adult F1and F2generations. Pyrosequencing was used to analyse the methylation level of paternally expressed imprinted genes. Our results showed that (1) For the8male reproduction-related non-imprinted genes, only down-regulated genes in newborn mice retained their differential expression pattern in adult F1generation, these genes were also down-regulated in IVF F1generation. Kcnqlot1, Plagl1, Snrpn, Peg3and Mest showed lower expression and higher methylation level in ICSI group than naturally conceived (NC) group, and the same changes were also shown in IVF mice. Compared with NC group, the changes on gene expression and DNA methylation level in IVF group was similar to that in ICSI group (2) Comparison between NC, COH, IVF and ICSI mice was done to analyze the effect of superovulation, in vitro culture and mechanical stimulation on the gene expression of testes. The down regulation of paternal expressed imprinted genes was mainly resulted from in vitro culture and mechanical stimulation. These three factors had an accumulative effect on the down regulation of male reproduction-related non-imprinted genes.(2) The differences of imprinted genes in ART F1mice were mostly observed in F2generation. The expression alteration of non-imprinted genes in ART Fl generation was mostly found in ICSI F2generations, but not in IVF F2generations.Conclusion:ICSI treatment can induce the alteration of differential gene expression in testes of offspring. The differential expression of genes in ICSI newborn mice can be persistent to adulthood, especially the changes in somatic cell of testis. Superovulation, in vitro culture and mechanical stimulation by ICSI were to some extent in charge of the differences of gene expression in testes. The alteration of DNA methylation modification was associated with the gene expressed difference in the testis of ART mice. ART treatments could induce the intergenerational transmission of differential gens expression, especially in ICSI group. Part II Alteration of fatty acid metabolism in ART male mice and the mechanism involvedLipid metabolism plays an important role in the whole process of pregnancy. Pathological abnormalities of assisted reproductive technology (ART) placentas have also been described, including abnormal placental shape, abnormal umbilical cord insertion and increased placental weights. Meanwhile, ART-induced impairment of placental steroid metabolism and restricted delivery of steroid hormones from mother to fetus have been reported. Our previous studies found a higher birth weight in ART mice. Therefore, we continued the topic of the health of ART male offspring and focused on the fatty acid metabolism in the second part. Moreover, the association between hormone and lipid metabolism or body weight is generally accepted, and the effect of superovulation on early embryos were also analyzed in this part to find the probable causes of gonadotropin induced abnormal lipid metabolism in offspring.Firstly we measured the fatty acid composition and the gene expression of lipogenesis and lipolysis-related enzymes in the liver and adipose tissue male mice produced by controlled ovarian hyperstimulation (COH), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The effect of the activities of fatty acid metabolism-related enzymes on fatty acid composition was estimated using the correlation analysis. Secondly, the mice were superovulated with two doses of gonadotropin, and the fertilization rate, lipid content and distribution, fatty acid composition and the expression of the triglyceride metabolism-related genes in embryos was analyzed. Moreover, the content of ovarian cholesterol and serum progesterone were also detected to analyze their effect on lipid metabolism of embryos.Our results showed that (1) The significant alteration in the liver and adipose tissue compared to naturally conceived (NC) mice. Compared with the NC mice, ART mice have significantly lower level of Polyunsaturated fatty acids (PUFAs) and higher level of Saturated fatty acids (SFAs) and Monounsaturated fatty acids (MUFAs), while the alteration of fatty acid profile in the liver of ART mice was much more complex. The difference of fatty acid composition between the four groups of mice varied with age.(2)Using correlation analysis, we found lipid metabolism-related enzymes may influence fatty acid composition by regulating the content of C16:0and C18:0in liver and adipose tissue respectively. Compared with NC group, the expressions of lipogenic and lipolytic genes altered in ART groups, and the expressed changes of these genes with age were different between ART and NC group. The expression of fatty acid synthesis genes in ART groups showed the same changing trend compared with NC group in adipose tissue of both adult and old mice.(3) Gonadotropin increased lipid accumulation and decreased fatty acid synthesis in a dose-dependent manner, and the changes of fatty acid composition were also shown in gonadotropin-induced embryos.These results indicated that ART treatments might have effect on the fatty acid composition and the expression of fatty acid metabolism-related genes in adipose tissue and liver of male offspring. The alteration of fatty acid composition was associated with ART induced alterations of activities and expressions of fatty acid metabolism-related enzymes. The changing trend of SFAs, MUFAs, PUFAs and the expression of fatty acid synthesis genes was consistent in adipose tissue of COH, IVF and ICSI mice, which suggested the effect of gonadotropin might be an key factor in the abnormal fatty acid metabolism of ART offspring. The effect of gonadotropin on lipid metabolism in offspring might be originated from the early embryo development.