Ruixiu Zhang, Lu Wang, Hexia Xia and Wei Zhang
Naihong Yan, Yilu Lu, Huaqin Sun, Dachang Tao, Sizhong Zhang, Wenying Liu and Yongxin Ma
MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Recent studies indicate that miRNAs are mechanistically involved in the development of mammalian spermatogenesis. However, little work has been done to compare the miRNA expression patterns between immature and mature mouse testes. Here, we employed a miRNA microarray to detect 892 miRNAs in order to evaluate the expression patterns of miRNA. The expression of 19 miRNAs was significantly different between immature and mature individuals. Fourteen miRNAs were significantly upregulated and five miRNAs were downregulated in immature mice and this result was further confirmed by a quantitative real-time RT-PCR assay. Many target genes involved in spermatogenesis are predicted by MiRscan performing miRNA target scanning. Our data indicated specific miRNAs expression in immature mouse testis and suggested that miRNAs have a role in regulating spermatogenesis.
Xiaoyan Huang, Jun Zhang, Li Lu, Lanlan Yin, Min Xu, Youqun Wang, Zuomin Zhou and Jiahao Sha
Identification of genes specifically expressed in adult and fetal testis is important in furthering our understanding of testis development and function. In this study, a novel human transcript, designated human testis cAMP-responsive element-binding protein (htCREB), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray containing 9216 clones. The htCREB transcript (GenBank Accession no. AY347527) was expressed at 2.35-fold higher levels in adult human testes than in fetal testes. Sequence and ntBLAST analyses against the human genome database indicated that htCREB was a novel splice variant of human CREB. RT-PCR-based tissue distribution experiments demonstrated that the htCREB transcript was highly expressed in adult human testis and in healthy sperm, but not in testes from patients with Sertoli cell-only syndrome. Taken together, these results suggest that the htCREB transcript is chiefly expressed in germ cells and is most likely involved in spermatogenesis.
Min An, Dong Li, Ming Yuan, Qiuju Li, Lu Zhang and Guoyun Wang
Endometrial cells and microenvironment are two important factors in the pathogenesis of adenomyosis. Our previous study demonstrated that macrophages can induce eutopic epithelial cells of adenomyosis to suffer from epithelial–mesenchymal transition (EMT). The aim of this study is to detect whether macrophages interacting with epithelial cells equally induce the EMT process in normal and eutopic endometria of healthy and adenomyotic patients; and whether macrophages parallelly polarize to M2. We investigated the expression levels of epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), cytokeratin7 (CK7), vimentin, transforming growth factor-β1 (TGFB1), SMAD3 and pSMAD3 using immunohistochemistry and western blot, and then estimated the genetic levels of CD163, IL10 and MMP12 using real-time quantitative polymerase chain reaction (RT-PCR) in macrophages. Eutopic and normal endometrial tissues were obtained from 20 patients with adenomyosis and 11 control patients without adenomyosis, respectively. The immunohistochemical analysis shows distinct EMT in eutopic endometria in secretory phase; the expression levels of TGFB1, SMAD3 and pSMAD3 that indicate signal pathway of EMT were also higher in secretory phase. Macrophages can induce EMT process in primary endometrial epithelial cells derived from normal and eutopic endometria. After co-culturing, THP-1-derived macrophages polarized to M2. Compared with the eutopic endometrium group, further polarization to M2 was observed in the normal endometrium group. These results indicate that adenomyosis may be promoted by the pathologic EMT of epithelial cells, which is induced by macrophages that incapably polarize to M2.
Ruizhi Deng, Chengquan Han, Lu Zhao, Qing Zhang, Beifen Yan, Rui Cheng, Biao Wei, Peng Meng, Tingchao Mao, Yong Zhang and Jun Liu
Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.
Wen-Lin Chang, Qing Yang, Hui Zhang, Hai-Yan Lin, Zhi Zhou, Xiaoyin Lu, Cheng Zhu, Li-Qun Xue and Hongmei Wang
Placenta-specific protein 1 (PLAC1), a placenta-specific gene, is known to be involved in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration are investigated by technical studies including real-time RT-PCR, in situ hybridization, immunohistochemistry, and functional studies by utilizing cell invasion and migration assays in the trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblasts (EVTs). The results show that PLAC1 is mainly detected in the trophoblast columns and syncytiotrophoblast of the first-trimester human placental villi, as well as in the EVTs that invade into the maternal decidua. Knockdown of PLAC1 by RNA interference significantly suppresses the invasion and migration of HTR8/SVneo cells and shortens the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the above data suggests that PLAC1 plays an important role in human placental trophoblast invasion and migration.
Zhenzhen Zhang, Changjiu He, Lu Zhang, Tianqi Zhu, Dongying Lv, Guangdong Li, Yukun Song, Jing Wang, Hao Wu, Pengyun Ji and Guoshi Liu
α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.
Ziliang Ji, Ruijing Lu, Lisha Mou, Yong-Gang Duan, Qiang Zhang, Yadong Wang, Yaoting Gui and Zhiming Cai
Hyperthermia and oxidative stresses are the two central elements contributing to varicocele-related sperm damage. Growing evidence indicates that microRNAs (miRNAs) are involved in the regulation of the heat and oxidative stress responses. In this study, we analyzed the expressions of several stress-related miRNAs in the sperm and found that the expression of miR-15a was significantly decreased in patients with varicocele compared with the control. Furthermore, miR-15a repressed the expression of HSPA1B, which is a typical stress-induced chaperone protein, through directly binding its 3′-UTR. The expressions of miR-15a and HSPA1B exhibited an inverse correlation in sperm. Our results provide a valuable insight into the varicocele-related sperm impairment and male infertility, and may help to develop potential therapeutic targets and novel biomarkers for male infertility.
Guangdong Li, Xiuzhi Tian, Dongying Lv, Lu Zhang, Zhenzhen Zhang, Jing Wang, Minghui Yang, Jingli Tao, Teng Ma, Hao Wu, Pengyun Ji, Yingjie Wu, Zhengxing Lian, Wei Cui and Guoshi Liu
NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.