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Lifan Zhang, Xing Du, Shengjuan Wei, Dongfeng Li and Qifa Li

As a key mediator of the transforming growth factor-beta (TGF-β) signaling pathway, which plays a pivotal role in regulating mammalian reproductive performance, Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs in SMAD4-siRNA treated cells were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-β signaling pathway were discovered as the canonical pathways changed under SMAD4-silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs, thus improving our understanding of regulatory mechanisms of SMAD4 gene in follicular development.

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Xiangyun Li, Wei Wei, Jun Yong, Qing Jia, Yuansong Yu and Keqian Di

The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

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Jing Tong, Shile Sheng, Yun Sun, Huihui Li, Wei-Ping Li, Cong Zhang and Zi-Jiang Chen

Good-quality oocytes are critical for the success of in vitro fertilization (IVF), but, to date, there is no marker of ovarian reserve available that can accurately predict oocyte quality. Melatonin exerts its antioxidant actions as a strong radical scavenger that might affect oocyte quality directly as it is the most potent antioxidant in follicular fluid. To investigate the precise role of endogenous melatonin in IVF outcomes, we recruited 61 women undergoing treatment cycles of IVF or intracytoplasmic sperm injection (ICSI) procedures and classified them into three groups according to their response to ovarian stimulation. Follicular fluid was collected to assess melatonin levels using a direct RIA method. We found good correlations between melatonin levels in follicular fluid with age, anti-Müllerian hormone (AMH) and baseline follicle-stimulating hormone (bFSH), all of which have been used to predict ovarian reserve. Furthermore, as melatonin levels correlated to IVF outcomes, higher numbers of oocytes were collected from patients with higher melatonin levels and consequently the number of oocytes fertilized, zygotes cleaved, top quality embryos on D3, blastocysts obtained and embryos suitable for transplantation was higher. The blastocyst rate increased in concert with the melatonin levels across the gradient between the poor response group and the high response group. These results demonstrated that the melatonin levels in follicular fluid is associated with both the quantity and quality of oocytes and can predict IVF outcomes as well making them highly relevant biochemical markers of ovarian reserve.

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Mei-rong Zhao, Wei Qiu, Yu-xia Li, Zhi-bin Zhang, Dong Li and Yan-ling Wang

Transforming growth factor β (TGFβ) has been shown to be a multifunctional cytokine required for embryonic development and regulation of trophoblast cell behaviors. In the present study, a non-transformed cell-line representative of normal human trophoblast (NPC) was used to examine the effect of TGFβ1 on trophoblast cell adhesion and invasion. In vitro assay showed that TGFβ1 could significantly promote intercellular adhesion, while inhibiting cell invasion across the collagen I-coated filter. Reverse transcription (RT)-PCR and gelatin zymography demonstrated that TGFβ1 evidently repressed the mRNA expression and proenzyme production of matrix metalloproteinase (MMP)-9, but exerted no effect on mRNA expression and secretion of MMP-2. On the other hand, both the mRNA and protein expression of epithelial-cadherin and β-catenin were obviously upregulated by TGFβ1 in dose-dependent fashion, as revealed by RT-PCR and western-blot analysis. What is more, one of the critical TGFβ signaling molecules – Smad2 was notably phosphorylated in TGFβ1-treated NPC cells. The data indicates that cell invasion and adhesion are coordinated processes in human trophoblasts and that there exists paracrine regulation on adhesion molecules and invasion-associated enzymes in human placenta.

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Yong-Hai Li, Yi Hou, Wei Ma, Jin-Xiang Yuan, Dong Zhang, Qing-Yuan Sun and Wei-Hua Wang

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm–oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm–oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 ± 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 ± 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm–oocyte interactions during fertilization.

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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.

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Wei Wang, Xia Chen, Xinxiu Li, Li Wang, Haiyan Zhang, Yu He, Jingjing Wang, Yongyan Zhao, Baole Zhang and Yinxue Xu

FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor β (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.

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Shun Zhang, Yi Zeng, Juan Qu, Yaning Luo, Xiaohong Wang and Wei Li

Administration of exogenous epidermal growth factor (EGF) improves testicular injury after acute ischemia–reperfusion (IR) stress, but the molecular basis is poorly understood. The role of endogenous EGF in testicular recovery and the underlying intracellular signaling pathways involved were herein investigated. In mice, testicular IR injury significantly enhanced the expression level of endogenous Egf at the very beginning of reperfusion. Expression of EGF receptor (Egfr (ErbB1)) was accordingly upregulated 3 h after reperfusion. Deprivation of majority of circulated EGF by sialoadenectomy aggravated testicular detriment (especially in pachytene spermatocytes), enhanced germ cell apoptosis, and thereafter resulted in impaired meiotic differentiation after IR insult. Mechanistically, endogenous EGF signaling appeared to be indispensable for the proper maintenance of Sertoli germ cells anchoring junction dynamics during the early testicular recovery. We also provided the in vitro evidences in a well-established rat Sertoli germ cell co-cultures model that the pro-survival effect of endogenous EGF on germ cells in response to testicular IR insult is mediated, at least in part, via the phosphatidylinositol 3-kinase/pAkt pathway. Collectively, our results suggest that the augment of endogenous EGF during the early testicular recovery may act on top of an endocrinous cascade orchestrating the intimate interactions between Sertoli cells and germ cells and may operate as indispensable defensive mechanism in response to testicular IR stress. Future studies in this field would shed light on this complicated pathogenesis.

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Xiu Shi, Wei Xu, Hui-Hua Dai, Ying Sun and Xiu-Li Wang

To compare the expression patterns of steroid receptor coactivators (SRCs) and steroid-induced stromal cell-derived factor 1 (CXCL12 (SDF1)) in normal and ectopic endometrium and to explore the roles of NCOA1 (SRC1) and NCOA2 (SRC2) in the steroid-induced CXCL12 expression in normal and ectopic endometrial stromal cells (ESCs). The NCOA1, NCOA2, NCOA3 (SRC3), and CXCL12 (SDF1)α mRNA levels in normal and ectopic endometrium were analyzed by quantitative real-time PCR. Steroid-induced CXCL12 expression was detected by the ELISA method and the chemotactic activity of conditioned supernatant to monocyte was assessed by the Boyden chamber method before and after the silencing of NCOA1 or NCOA2 with siRNA in normal and ectopic ESCs. The expression of NCOA1 and CXCL12 in ectopic endometrium was significantly greater than that in normal endometrium in the secretory phase. Progesterone (P4) was able to significantly inhibit estradiol (E2)-stimulated CXCL12 expression in normal and ectopic ESCs. The inhibitory rate of P4 in ectopic ESCs at 72 and 96 h was significantly lower than that in normal ESCs. Silencing of NCOA1 but not NCOA2 significantly reduced the E2-induced CXCL12 expression in normal and ectopic ESCs. The ability of P4 to inhibit E2-induced CXCL12 expression and monocyte chemotaxis in normal and ectopic ESCs was significantly attenuated when NCOA2 was silenced. NCOA1 plays a necessary role in E2-induced CXCL12 expression and NCOA2 is required for P4 to inhibit the E2-induced CXCL12 production in normal and ectopic endometrium.

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Chao Wei, Xia Li, Pengfei Zhang, Yu Zhang, Tong Liu, Shaoshuai Jiang, Fei Han and Yunhai Zhang

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos.

Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/149/5/485/suppl/DC2.