Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos.

During the first lineage segregation, a mammalian totipotent embryo differentiates into the inner cell mass (ICM) and trophectoderm (TE). However, how transcription factors (TFs) regulate this earliest cell-fate decision in vivo remains elusive, with their regulomes primarily inferred from cultured cells. Here, we investigated the TF regulomes during the first lineage specification in early mouse embryos, spanning the pre-initiation, initiation, commitment, and maintenance phases. Unexpectedly, we found that TFAP2C, a trophoblast regulator, bound and activated both early TE and inner cell mass (ICM) genes at the totipotent (two- to eight-cell) stages ('bipotency activation'). Tfap2c deficiency caused downregulation of early ICM genes, including Nanog, Nr5a2, and Tdgf1, and early TE genes, including Tfeb and Itgb5, in eight-cell embryos. Transcription defects in both ICM and TE lineages were also found in blastocysts, accompanied by increased apoptosis and reduced cell numbers in ICMs. Upon trophoblast commitment, TFAP2C left early ICM genes but acquired binding to late TE genes in blastocysts, where it co-bound with CDX2, and later to extra-embryonic ectoderm (ExE) genes, where it cooperatively co-occupied with the former ICM regulator SOX2. Finally, 'bipotency activation' in totipotent embryos also applied to a pluripotency regulator NR5A2, which similarly bound and activated both ICM and TE lineage genes at the eight-cell stage. These data reveal a unique transcription circuity of totipotency underpinned by highly adaptable lineage regulators.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

NR5A2 connects zygotic genome activation to the first lineage segregation in totipotent embryos.

Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to the inner cell mass (ICM) where pluripotent epiblast arises, and extraembryonic trophectoderm. However, how ZGA is connected to the first lineage segregation in mammalian embryos remains elusive. Here, we investigated the role of nuclear receptor (NR) transcription factors (TFs), whose motifs are highly enriched and accessible from the 2-cell (2C) to 8-cell (8C) stages in mouse embryos. We found that NR5A2, an NR TF strongly induced upon ZGA, was required for this connection. Upon Nr5a2 knockdown or knockout, embryos developed beyond 2C normally with the zygotic genome largely activated. However, 4-8C-specific gene activation was substantially impaired and Nr5a2-deficient embryos subsequently arrested at the morula stage. Genome-wide chromatin binding analysis showed that NR5A2-bound cis-regulatory elements in both 2C and 8C embryos are strongly enriched for B1 elements where its binding motif is embedded. NR5A2 was not required for the global opening of its binding sites in 2C embryos but was essential to the opening of its 8C-specific binding sites. These 8C-specific, but not 2C-specific, binding sites are enriched near genes involved in blastocyst and stem cell regulation, and are often bound by master pluripotency TFs in blastocysts and embryonic stem cells (ESCs). Importantly, NR5A2 regulated key pluripotency genes Nanog and Pou5f1/Oct4, and primitive endoderm regulatory genes including Gata6 among many early ICM genes, as well as key trophectoderm regulatory genes including Tead4 and Gata3 at the 8C stage. By contrast, master pluripotency TFs NANOG, SOX2, and OCT4 targeted both early and late ICM genes in mouse ESCs. Taken together, these data identify NR5A2 as a key regulator in totipotent embryos that bridges ZGA to the first lineage segregation during mouse early development.

OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the 'preconfiguration' of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation.

Translational regulation plays a critical role during the oocyte-to-embryo transition (OET) and zygotic genome activation (ZGA). Here, we integrated ultra-low-input ribosome profiling (Ribo-lite) with messenger RNA sequencing to co-profile the translatome and transcriptome in human oocytes and early embryos. Comparison with mouse counterparts identified widespread differentially translated gene functioning in epigenetic reprogramming, transposon defense, and small RNA biogenesis, in part driven by species-specific regulatory elements in 3' untranslated regions. Moreover, PRD-like homeobox transcription factors, including TPRXL, TPRX1, and TPRX2, are highly translated around ZGA. TPRX1/2/L knockdown leads to defective ZGA and preimplantation development. Ectopically expressed TPRXs bind and activate key ZGA genes in human embryonic stem cells. These data reveal the conservation and divergence of translation landscapes during OET and identify critical regulators of human ZGA.

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA.

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

Phosphatidylcholine could protect the defect of zearalenone exposure on follicular development and oocyte maturation.

Zearalenone (ZEA) is a well-known exogenous endocrine disruptor and can lead to severe negative effects on the human and animal reproductive process. Using a follicle culture model, we have previously shown that ZEA exposure significantly affected the follicular development and antrum formation but the underlying mechanisms are not well known. Therefore, in this study, we explored the metabolomic changes of granulosa cell (GC) culture media with or without ZEA exposure. The results showed that ZEA significantly increased phosphatidylcholine or phosphatidyl ethanolamine adducts in culture medium. A comprehensive analysis with the metabolome data from follicular fluid of small and large antral follicles showed that lyso phosphatidylcholine (LPC) was accumulated during follicle growth, but was depleted by ZEA exposure. Exogenous supplement with LPC to the follicle growth media or oocyte maturation media can partly protect the defect of ZEA exposure on follicular antrum formation and oocyte maturation. Taken together, our results demonstrate that ZEA exposure hinders the follicular growth and exogenous LPC can practically protect the defect of ZEA on follicular development and oocyte maturation.

Analysis of the SNP loci around transcription start sites related to goat fecundity trait base on whole genome resequencing.

Genome flanking regions surrounding transcription start sites (TSSs) are critical for the regulation of gene expression, containing many translational regulatory elements. To investigate whether critical single nucleotide polymorphisms (SNPs) exist around TSSs in the dairy goat genome, we performed high throughput DNA sequencing to compare two dairy goat groups with discrepant litter sizes. After genome mapping, SNP calling, and annotation, we screened the SNPs within 2kb scales surrounding annotated TSSs in high fecundity (HF) and low fecundity (LF) groups, respectively. We attempted to identify distinct SNPs and motifs near the TSSs in both groups. The SNPs near the TSSs most were consistent; 318 new SNPs were uncovered in the HF group, of which 305 were heterozygote SNPs, 13 were homozygote SNPs, and majority of which were distributed on chromosome 2 and 29. After validation by Sanger sequencing we found that a SNP in CHI16: 27612330 C>A in the PSEN2 gene presented an A/A genotype in the HF group and an A/A or A/C genotype in the LF group. In conclusion, our study provides insightful information into the dairy goat genomic variations surrounding TSSs, which may contribute to enhanced litter size. Based on comparison studies of SNPs exist around transcription start sites between high fecundity group and low fecundity group. Our finding provides insights concerning the goat litter size phenotypic and will promote future goat breeding.

LncRNA as ceRNAs may be involved in lactation process.

The main function of the mammary gland is to secret milk for newborn growth. Milk production process is regulated by hormones, growth factors, noncoding RNAs and other factors locally. Long non-coding RNAs (lncRNAs), one type of recently discovered non-coding RNA, have been found in mammary gland and some studies suggested lncRNA may play important roles in mammary gland development. Competing endogenous RNAs (ceRNAs) are emerging to compete for miRNA binding and, in turn, regulate each other. In the current study, we sequenced mRNA, miRNA and lncRNA in goat mammary tissue at 2 points in lactation (early and mature). All data were co-expressed together from the same samples. Our data showed that the ceRNAs up-regulated during the mature lactation phase were associated with lipid, protein, carbon and amino acid synthesis and metabolism. This correlates with the function of the mature lactation phase: i.e. the continuous production of large amounts of milk, rich in proteins, lipids, amino acids and other nutrients. Alternately, the ceRNAs up-regulated during early lactation were associated with PI3K-AKT pathways and ECM-receptor interactions; these fulfil the functional role of preparing the mammary gland for full lactation. Therefore, the results suggest that ceRNAs work synergistically during different developmental stages to regulate specific functions associated with lactation control. This study suggests that ceRNAs (lncRNA-mRNA) may be involved in lactation process.

Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro.

Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 µM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development.

Alleviation of Rosup-induced oxidative stress in porcine granulosa cells by anthocyanins from red-fleshed apples.

Anthocyanins are the polyphenolic phytochemicals which have been shown to scavenge free radicals. In this study, we investigated the effects of anthocyanins extracted from red-fleshed apples (Malus sieversii) on reducing oxidative damage by Rosup in porcine granulosa cells (GCs) by measuring intracellular reactive oxygen species (ROS), content of glutathione (GSH), activities of superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase (GPX1) and the gene expression of SOD1, CAT, GPX1. Apoptosis was determined with TdT-mediated dUTP-biotin nick end labeling (TUNEL) and apoptosis-related proteins were quantified with Western blotting. The results indicate that Rosup increases oxidative stress by inducing reactive oxygen species production in porcine GCs and the oxidative stress could be reduced by anthocyanins. The gene expression of SOD1, CAT, GPX1 and the activities of these enzymes were increased when GCs were treated with anthocyanins and Rosup for 6 hours. Anthocyanins inhibit Rosup-induced apoptosis by increasing expression of antiapoptotic protein Bcl-2 and suppressing the expression of pro-apoptotic protein Bax. Collectively, anthocyanins from red-fleshed apples reduce oxidative stress and inhibit apoptosis in porcine GCs in vitro. This approach indicates that antioxidants might be developed from red-fleshed apples.

Cutaneous applied nano-ZnO reduce the ability of hair follicle stem cells to differentiate.

The ability of metal oxide nanoparticles to penetrate the skin has aroused a great deal of interest during the past decade due to concerns over the safety of topically applied sunscreens that contain physical UV-resistant metal particles, such as nano-Zinc oxide (nZnO). Previous studies demonstrate that metal oxide nanoparticles accumulate in skin furrows and hair follicles following topical application while little is known about the consequence of these nanoparticles on skin homeostasis. The current investigation tested the effects of nZnO (0.5 mg/day mouse) on hair follicle physiology. Topical application of Vaseline containing nZnO, bulk ZnO (bZnO), or ionized Zn to newborn mice vibrissa pad over a period of 7 consecutive days revealed that nZnO accumulated within hair follicles, and this induced the apoptosis of hair follicle stem cells (HFSCs). In vitro studies also indicated that nZnO exposure caused obvious DNA damage and induced apoptosis in HFSCs. Furthermore, it was found that nZnO exposure perturbed genes associated with HFSC apoptosis, cell communication, and differentiation. HFSCs transplantation assay demonstrated that the potential of HFSCs to differentiate was reduced. This investigation indicates a potential risk of topically applied ZnO nanoparticles on skin homeostasis.

Di (2-ethylhexyl) phthalate impairs steroidogenesis in ovarian follicular cells of prepuberal mice.

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer which is widely used in the manufacture of plastics. As a common environmental contaminant and recognized endocrine disrupting chemical, DEHP is able to deregulate the functions of a variety of tissues, including the reproductive system both in males and females. In order to investigate the possible effects of DEHP on the first wave of folliculogenesis, occurring in the mouse ovary postnatally, mice were administered 20 or 40 µg/kg DEHP through intraperitoneal injection at days 5, 10 and 15 post partum (dpp). Following DEHP treatment the gene expression profile of control and exposed ovaries was compared by microarray analyses at 20 dpp. We found that in the exposed ovaries DEHP significantly altered the transcript levels of several immune response and steroidogenesis associated genes. In particular, DEHP significantly decreased the expression of genes essential for androgen synthesis by theca cells including Lhcgr, Cyp17a1, Star and Ldlr. Immunohistochemistry and immune flow cytometry confirmed reduced expression of LHCGR and CYP17A1 proteins in the exposed theca cells. These effects were associated to a significant reduction in ovarian concentrations of progesterone, 17ß-estradiol and androstenedione along with a reduction of LH in the serum. Although we did not find a significant reduction of the number of primary, secondary or antral follicles in the DEHP exposed ovaries when compared to controls, we did observe that theca cells showed an altered structure of the nuclear envelope, fewer mitochondria, and mitochondria with a reduced number of cristae. Collectively, these results demonstrate a deleterious effect of DEHP exposure on ovarian steroidogenesis during the first wave of folliculogenesis that could potentially affect the correct establishment of the hypothalamic-pituitary-ovarian axis and the onset of puberty.

Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus).

Dairy goats are one of the most utilized domesticated animals in China. Here, we selected extreme populations based on differential fecundity in two Laoshan dairy goat populations. Utilizing deep sequencing we have generated 68.7 and 57.8 giga base of sequencing data, and identified 12,458,711 and 12,423,128 SNPs in the low fecundity and high fecundity groups, respectively. Following selective sweep analyses, a number of loci and candidate genes in the two populations were scanned independently. The reproduction related genes CCNB2, AR, ADCY1, DNMT3B, SMAD2, AMHR2, ERBB2, FGFR1, MAP3K12 and THEM4 were specifically selected in the high fecundity group whereas KDM6A, TENM1, SWI5 and CYM were specifically selected in the low fecundity group. A sub-set of genes including SYCP2, SOX5 and POU3F4 were localized both in the high and low fecundity selection windows, suggesting that these particular genes experienced strong selection with lower genetic diversity. From the genome data, the rare nonsense mutations may not contribute to fecundity, whereas nonsynonymous SNPs likely play a predominant role. The nonsynonymous exonic SNPs in SETDB2 and CDH26 which were co-localized in the selected region may take part in fecundity traits. These observations bring us a new insights into the genetic variation influencing fecundity traits within dairy goats.

Di (2-ethylhexyl) Phthalate Exposure Impairs Growth of Antral Follicle in Mice.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 µg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.

The influence of N-acetyl-l-cysteine on damage of porcine oocyte exposed to zearalenone in vitro.

Zearalenone (ZEA), one of the mycotoxins produced by Fusarium fungi, impacts porcine reproduction by interfering with the estrogen signaling pathway. Previous studies have shown that ZEA inhibits porcine oocyte maturation through the formation of aberrant spindle. To explore the effect of ZEA on porcine oocyte meiotic maturation, the extent of both nuclear and cytoplasmic maturation was examined in this study. Compared with control group, presence of ZEA (3 µM) during oocyte maturation, significantly inhibited the polar body extrusions from 71% to 51%, and significantly increased intracellular reactive oxygen species (ROS) level (12.01 vs. 5.89). Intracellular glutathione (GSH) content in ZEA treatment group was lower than in the control group (1.08 pmol/oocyte vs. 0.18 pmol/oocyte), and cortical granules of cortical area distributed oocytes were reduced (88% vs. 62%). ZEA decreases cumulus expansion in both morphology and mRNA level (HAS2, PTX3, TNFAIP6 and CX43). Addition of N-acetyl-l-cysteine (NAC) to the oocyte maturation media reversed the ZEA-induced inhibition of polar body extrusion (from 69% to 81%), up-regulated ROS (from 7.9 to 6.5), down-regulated GSH content (from 0.16 to 0.82 pmol/oocyte) and recovered cumulus cells expansion in morphology and mRNA level. It is concluded that ZEA affects both oocyte nucleus and cytoplasmic maturation during in vitro maturation, and NAC can reverse these damages to some extent.

Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse.

The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5-3.5 days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice.

Oxidative stress induced by zearalenone in porcine granulosa cells and its rescue by curcumin in vitro.

Oxidative stress (OS), as a signal of aberrant intracellular mechanisms, plays key roles in maintaining homeostasis for organisms. The occurrence of OS due to the disorder of normal cellular redox balance indicates the overproduction of reactive oxygen species (ROS) and/or deficiency of antioxidants. Once the balance is broken down, repression of oxidative stress is one of the most effective ways to alleviate it. Ongoing studies provide remarkable evidence that oxidative stress is involved in reproductive toxicity induced by various stimuli, such as environmental toxicants and food toxicity. Zearalenone (ZEA), as a toxic compound existing in contaminated food products, is found to induce mycotoxicosis that has a significant impact on the reproduction of domestic animals, especially pigs. However, there is no information about how ROS and oxidative stress is involved in the influence of ZEA on porcine granulosa cells, or whether the stress can be rescued by curcumin. In this study, ZEA-induced effect on porcine granulosa cells was investigated at low concentrations (15 µM, 30 µM and 60 µM). In vitro ROS levels, the mRNA level and activity of superoxide dismutase, glutathione peroxidase and catalase were obtained. The results showed that in comparison with negative control, ZEA increased oxidative stress with higher ROS levels, reduced the expression and activity of antioxidative enzymes, increased the intensity of fluorogenic probes 2', 7'-Dichlorodihydrofluorescin diacetate and dihydroethidium in flow cytometry assay and fluorescence microscopy. Meanwhile, the activity of glutathione (GSH) did not change obviously following 60 µM ZEA treatment. Furthermore, the underlying protective mechanisms of curcumin on the ZEA-treated porcine granulosa cells were investigated. The data revealed that curcumin pre-treatment significantly suppressed ZEA-induced oxidative stress. Collectively, porcine granulosa cells were sensitive to ZEA, which may induce oxidative stress. The findings from this study clearly demonstrate that curcumin is effective to reduce the dysregulation of cellular redox balance on porcine granulosa cells in vitro and should be further investigated for its protective role against ZEA in animals.