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Quanwei Wei, Wei Ding and Fangxiong Shi

Poly(ADP-ribosylation), which occurs rapidly in cells following DNA damage and is regulated by poly (ADP-ribose) polymerase 1 (PARP1), is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. Although PARP1 has recently been implicated in a variety of physiological and pathological processes, its role in the process of follicular development and atresia is not yet completely defined. This study was designed to investigate the cellular expression pattern and immunolocalization of PARP1, cleaved PARP1, caspase 3, and cleaved caspase 3 in fetal, neonatal, and adult porcine ovaries. Our results showed that in fetal and neonatal pigs, PARP1 cleavage is involved in the process of oocyte nest breakdown, primordial follicle formation, and transition to primary follicles. The results of immunohistochemistry indicated that PARP1 cleavage was involved in the process of follicular development and atresia, which was in accordance with our previous study; however, it was noted that cleaved caspase 3 was mainly localized in and around the nucleus of apoptotic granulosa cells (GCs), whereas cleaved PARP1 was mainly localized in the nucleus of the apoptotic GCs. RIA data showed increased serum progesterone and estradiol concentrations with age after birth. Collectively, our findings suggest that the PARP1 signaling pathway is involved in oocyte nest breakdown and primordial follicle formation in fetal and neonatal porcine ovaries, but is different from follicular atresia in adult porcine ovaries that involves cellular apoptosis.

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Wei-Bin Wu, Yue-Ying Xu, Wei-Wei Cheng, Bo Yuan, Jiu-Ru Zhao, Yan-Lin Wang and Hui-Juan Zhang

Fetal growth restriction (FGR) threatens perinatal health and is correlated with increased incidence of fetal original adult diseases. Most cases of FGR were idiopathic, which were supposed to be associated with placental abnormality. Decreased circulating placental growth factor (PGF) was recognized as an indication of placental deficiency in FGR. In this study, the epigenetic regulation of PGF in FGR placentas and the involvement of PGF in modulation of trophoblast activity were investigated. The expression level of PGF in placental tissues was determined by RT-qPCR, immunohistochemistry and ELISA. DNA methylation profile of PGF gene was analyzed by bisulfite sequencing. Trophoblastic cell lines were treated with ZM-306416, an inhibitor of PGF receptor FLT1, to observe the effect of PGF/FLT1 signaling on cell proliferation and migration. We demonstrated that PGF was downregulated in placentas from FGR pregnancies compared with normal controls. The villous expression of PGF was positively correlated with placental and fetal weight. The CpG island inside PGF promoter was hypomethylated without obvious difference in both normal and FGR placentas. However, the higher DNA methylation at another CpG island downstream exon 7 of PGF was demonstrated in FGR placentas. Additionally, we found FLT1 was expressed in trophoblast cells. Inhibition of PGF/FLT1 signaling by a selective inhibitor impaired trophoblast proliferation and migration. In conclusion, our data suggested that the PGF expression was dysregulated, and disrupted PGF/FLT1 signaling in trophoblast might contribute to placenta dysfunction in FGR. Thus, our results support the significant role of PGF in the pathogenesis of FGR.

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Chelsea Marcho, Wei Cui and Jesse Mager

Successful mammalian development requires descendants of single-cell zygotes to differentiate into diverse cell types even though they contain the same genetic material. Preimplantation dynamics are first driven by the necessity of reprogramming haploid parental epigenomes to reach a totipotent state. This process requires extensive erasure of epigenetic marks shortly after fertilization. During the few short days after formation of the zygote, epigenetic programs are established and are essential for the first lineage decisions and differentiation. Here we review the current understanding of DNA methylation and histone modification dynamics responsible for these early changes during mammalian preimplantation development. In particular, we highlight insights that have been gained through next-generation sequencing technologies comparing human embryos to other models as well as the recent discoveries of active DNA demethylation mechanisms at play during preimplantation.

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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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Ruixiu Zhang, Lu Wang, Hexia Xia and Wei Zhang

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Zelieann R Craig, Wei Wang and Jodi A Flaws

Endocrine-disrupting chemicals (EDCs) are exogenous agents with the ability to interfere with processes regulated by endogenous hormones. One such process is female reproductive function. The major reproductive organ in the female is the ovary. Disruptions in ovarian processes by EDCs can lead to adverse outcomes such as anovulation, infertility, estrogen deficiency, and premature ovarian failure among others. This review summarizes the effects of EDCs on ovarian function by describing how they interfere with hormone signaling via two mechanisms: altering the availability of ovarian hormones, and altering binding and activity of the hormone at the receptor level. Among the chemicals covered are pesticides (e.g. dichlorodiphenyltrichloroethane and methoxychlor), plasticizers (e.g. bisphenol A and phthalates), dioxins, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons (e.g. benzo[a]pyrene).

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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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Wei Zhang, Sizhong Zhang, Cuiying Xiao, Yuan Yang and A Zhoucun

Fkbp6 has been proved to be a new component of synaptonemal complexes and be involved in homologous chromosomes pairing and male infertility in mice. To explore the possible association between variations in the FKBP6 gene and impaired spermatogenesis in human, mutation screening of all the eight exons and the intron/exon boundaries of the gene was performed in 323 patients with azoospermia or severe oligozoospermia and 205 fertile controls by denatured HPLC and DNA sequencing. As a result, four novel and one known single nucleotide transitions were identified, including c.58-2A>G, c.111C>T, c.156G>T, c.594G>A, and c.216C>A (rs3750075). The frequencies of genotype CA, allele A of c.216C>A and haplotype ‘GAG’ consisting of c.156G>T, c.216C>A, and c.594G>A were significantly lower in infertile patients than those in controls. These findings suggest that the FKBP6 gene may play a role in modifying the susceptibility to idiopathic spermatogenic impairment in human and propose that the allele A of c.216C>A seems to be a protective factor for the development of male infertility.

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Wei Si, Hongsheng Men, James D Benson and John K Critser

Osmotic stress is an important factor that can result in cell damage during cryopreservation. Before ejaculation or collection for cryopreservation, murine spermatozoa are stored in epididymal fluid, a physiologically hyperosmotic environment (∼415 mmol/kg). The objectives of this study were to determine the osmotic tolerance limits of sperm motion parameters of ICR and C57BL/6 mouse spermatozoa collected in isosmotic (290 mmol/kg) and hyperosmotic (415 mmol/kg) media, and the effect of the osmolality of sperm collection media on sperm fertility after cryopreservation. Our results indicate that murine spermatozoa collected in media with different osmolalities (290 and 415 mmol/kg Dulbecco's phosphate buffered saline (DPBS)) appeared to have different osmotic tolerances for the maintenance of sperm motility and other motion parameters in both mouse strains. The hypo- and hyperosmotic treatments decreased motility and affected other motion parameters of spermatozoa collected in 290 mmol/kg DPBS. The extent of the change of motion parameters after treatments corresponded with the levels of osmotic stress. However, for spermatozoa collected in 415 mmol/kg DPBS, exposure to 290 mmol/kg DPBS tended to increase sperm motility and the quality of their motion parameters. The osmolality of sperm collection medium can affect murine sperm fertility. Spermatozoa collected in 415 mmol/kg medium showed higher fertility compared with spermatozoa collected in 290 mmol/kg as assessed by IVF. Results characterizing murine sperm osmotic tolerance collected in media with different osmolalities from different strains and the effect of collection media osmolality on sperm fertility after cryopreservation will be useful in designing cryopreservation protocols.

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Fan Zhou, Wei Chen, Yiqun Jiang and Zuping He

Spermatogonial stem cells (SSCs) are one of the most significant stem cells with the potentials of self-renewal, differentiation, transdifferentiation and dedifferentiation, and thus, they have important applications in reproductive and regenerative medicine. They can transmit the genetic and epigenetic information across generations, which highlights the importance of the correct establishment and maintenance of epigenetic marks. Accurate transcriptional and post-transcriptional regulation is required to support the highly coordinated expression of specific genes for each step of spermatogenesis. Increasing evidence indicates that non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play essential roles in controlling gene expression and fate determination of male germ cells. These ncRNA molecules have distinct characteristics and biological functions, and they independently or cooperatively modulate the proliferation, apoptosis and differentiation of SSCs. In this review, we summarized the features, biological function and fate of mouse and human SSCs, and we compared the characteristics of lncRNAs and circRNAs. We also addressed the roles and mechanisms of lncRNAs and circRNAs in regulating mouse and human SSCs, which would add novel insights into the epigenetic mechanisms underlying mammalian spermatogenesis and provide new approaches to treat male infertility.