Hormone-sensitive lipase-knockout (HSL−/−) mice exhibit azoospermia for unclear reasons. To explore the basis of sterility, we performed the following three experiments. First, HSL protein distribution in the testis was determined. Next, transcriptome analyses were performed on the testes of three experimental groups. Finally, the fatty acid and cholesterol levels in the testes with three different genotypes studied were determined. We found that the HSL protein was present from spermatocyte cells to mature sperm acrosomes in wild-type (HSL+/+) testes. Spermiogenesis ceased at the elongation phase of HSL−/− testes. Transcriptome analysis indicated that genes involved in lipid metabolism, cell membrane, reproduction and inflammation-related processes were disordered in HSL−/− testes. The cholesterol content was significantly higher in HSL−/− than that in HSL+/+ testis. Therefore, gene expression and cholesterol ester content differed in HSL−/− testes compared to other testes, which may explain the sterility of male HSL−/− mice.
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Feng Wang, Zheng Chen, Xiaofang Ren, Ye Tian, Fucheng Wang, Chao Liu, Pengcheng Jin, Zongyue Li, Feixiong Zhang, and Baochang Zhu
Yanfang Wu, Zhenzi Zuo, Zheng Wang, Hanghang Liu, Qi Zhou, Subi Ren, Xinrui Lan, Yong Zhang, and Yongsheng Wang
Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer embryo. This paper reveals a need for us to further explore the roles of the paternal regulatory factors on embryonic development in early embryos.
Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidences show that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, cytoskeleton remodeling, embryonic development, and offspring phenotype. Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer (SCNT) embryo. bta-miR-183 was found to be highly expressed in bovine sperm and can be delivered into oocytes during fertilization in our previous study, and in this study, EZR was confirmed as a target gene of bta-miR-183 in early embryos by bioinformatics, luciferase, and gain-of-function and loss-of-function experiments. Scanning electron microscopy showed that the density of microvilli on the surface of SCNT embryos was significantly higher than that onin vitro fertilized embryos and was significantly reduced by injection of bta-miR-183 mimic. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that the lack of bta-miR-183 might lead to abnormal changes in microvilli by downregulating ezrin protein. In addition, gain-of-function studies showed that bta-miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, P < 0.05), blastocyst formation (43.75% vs 28.26%, P < 0.05), apoptotic index (5.21% vs 12.64%, P < 0.05), and the trophoblast ratio (32.65% vs 25.58%, P < 0.05) in day 7 blastocysts. Thus, the present study indicated that bta-miR-183 might play crucial roles in the formation of microvilli and embryo development by regulating expression of EZR mRNA.
Jiaojiao Huang, Hongyong Zhang, Jing Yao, Guosong Qin, Feng Wang, Xianlong Wang, Ailing Luo, Qiantao Zheng, Chunwei Cao, and Jianguo Zhao
Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14–16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.
Ru Zheng, Yue Li, Huiying Sun, Xiaoyin Lu, Bao-Fa Sun, Rui Wang, Lina Cui, Cheng Zhu, Hai-Yan Lin, and Hongmei Wang
The syncytiotrophoblast (STB) plays a key role in maintaining the function of the placenta during human pregnancy. However, the molecular network that orchestrates STB development remains elusive. The aim of this study was to obtain broad and deep insight into human STB formation via transcriptomics. We adopted RNA sequencing (RNA-Seq) to investigate genes and isoforms involved in forskolin (FSK)-induced fusion of BeWo cells. BeWo cells were treated with 50 μM FSK or dimethyl sulfoxide (DMSO) as a vehicle control for 24 and 48 h, and the mRNAs at 0, 24 and 48 h were sequenced. We detected 28,633 expressed genes and identified 1902 differentially expressed genes (DEGs) after FSK treatment for 24 and 48 h. Among the 1902 DEGs, 461 were increased and 395 were decreased at 24 h, whereas 879 were upregulated and 763 were downregulated at 48 h. When the 856 DEGs identified at 24 h were traced individually at 48 h, they separated into 6 dynamic patterns via a K-means algorithm, and most were enriched in down–even and up–even patterns. Moreover, the gene ontology (GO) terms syncytium formation, cell junction assembly, cell fate commitment, calcium ion transport, regulation of epithelial cell differentiation and cell morphogenesis involved in differentiation were clustered, and the MAPK pathway was most significantly regulated. Analyses of alternative splicing isoforms detected 123,200 isoforms, of which 1376 were differentially expressed. The present deep analysis of the RNA-Seq data of BeWo cell fusion provides important clues for understanding the mechanisms underlying human STB formation.
Ning Wang, Liya Wang, Fang Le, Qitao Zhan, Yingming Zheng, Guolian Ding, Xijing Chen, Jianzhong Sheng, Minyue Dong, Hefeng Huang, and Fan Jin
Despite the efforts to recapitulate the follicle environment, oocytes from in vitro maturation (IVM) have poorer developmental potential than those matured in vivo and the effects on the resultant offspring are of concern. The aim of this study was to determine altered gene expression in oocytes following IVM and to evaluate the expression of the arginine rich, mutated in early stage of tumors gene (Armet) and mitochondrial ribosomal protein L51 (Mrpl51) in embryos and brains of fetal/postnatal mice and the brain development of IVM offspring. An IVM mouse model was established while oocytes matured in vivo were used as the controls. Suppressive subtractive hybridization (SSH) and RT-PCR/western blot were used to analyze the differential expression of genes/proteins between IVM and the control group. HE staining and water maze were used to assess the histological changes in brain tissue and cognition of the offspring. The rates of fertilization, cleavage, and live birth were significantly decreased in IVM group. Thirteen genes were upregulated in IVM oocytes compared with the control, including Armet and Mrpl51. The higher level of Armet in IVM oocytes was retained in brain of newborn mice, which could be related to the upregulation of activating transcription factor 6 (Atf6) and X-box binding protein 1 (Xbp1), while Mrpl51 was expressed normally in brain of postnatal mice. No significant differences were detected in brain weight, neuronal counts, and the cognition in the offspring between the two groups. The present results suggested that IVM could affect the pregnancy outcome and the Armet and Mrpl51 gene/protein expression. The change in Armet expression lasted while the change of Mrpl51 disappeared after birth. However, the brain development of the offspring seemed to be unaffected by IVM.
Yue Li, Ru Zheng, Rui Wang, Xiaoyin Lu, Cheng Zhu, Hai-Yan Lin, Hongmei Wang, Xiaoguang Yu, and Jiejun Fu
The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.
Zheng-Bin Han, Guo-Cheng Lan, Yan-Guang Wu, Dong Han, Wei-Guo Feng, Jun-Zuo Wang, and Jing-He Tan
Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus–oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8–16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73–84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.
Kang Shan, Ma Xiao-Wei, Wang Na, Zhang Xiu-Feng, Wen Deng-Gui, Guo Wei, Zhang Zheng-Mao, and Li Yan
Endometriosis, one of the most frequent diseases in gynecology, is a benign but invasive and metastatic disease. The altered expression of E-cadherin may play an important role in developing endometriosis. In this paper, we discuss the association of three single nucleotide polymorphisms (SNPs) on the E-cadherin gene and risk of endometriosis. We examined the genotype frequency of three polymorphisms in 152 endometriosis patients and 189 control women. There was a significant difference in the frequency of the E-cadherin 3′-UTR C → T genotypes between endometriosis and controls (P = 0.01). The frequency of the C allele in patients (71.1%) was significantly higher than in the controls (63.8%; P = 0.04). When compared with the T/T + T/C genotypes, the C/C genotype had a significantly increased susceptibility to endometriosis, with an adjusted odds ratio of 1.79 (95% confidence interval = 1.17–2.76). No significant difference was found between endometriosis and control women on two polymorphisms (−160 C → A, −347 G → GA) at the gene promoter region of E-cadherin. The −160 C → A and −347 G → GA polymorphisms displayed linkage disequilibrium (D′ = 0.999). The −160 A/−347 GA haplotype was only detected in endometriosis patients (2%). These data show a relation between the E-cadherin 3′-UTR C → T polymorphism, the −160 A/−347 GA haplotype of two promoter polymorphisms and risk of endometriosis, suggesting a potential role in endometriosis development, at least in North Chinese women.
Wang Han-zheng, Lu Shu-hua, Shen Wei-xiong, Sun Zhi-da, Zhou Wei, Wu Yu-fen, and Zhou Mei-rong
Summary. Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase. Progesterone production was assessed after short-term incubation of luteal cell suspensions. Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0·01 i.u./ml, and the response was 5 times higher than the basal production.
Oestradiol (1–100 μm) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production. The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 μm. The ED50 values were approximately 3 μm, 75 μm and 100 μm for oestradiol, AF-45 and anordrin respectively. Anordrin showed no significant effects on basal progesterone production. In addition, oestradiol markedly inhibited the activity of 3β-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low.
The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.
Keywords: A-nor steroid; oestradiol; luteal cells; progesterone; 3β-hydroxysteroid dehydrogenase; man
Xue-Ying Zhang, Yi-Meng Xiong, Ya-Jing Tan, Li Wang, Rong Li, Yong Zhang, Xin-Mei Liu, Xian-Hua Lin, Li Jin, Yu-Ting Hu, Zhen-Hua Tang, Zheng-Mu Wu, Feng-Hua Yin, Zheng-Quan Wang, Ye Xiao, Jian-Zhong Sheng, and He-Feng Huang
Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.