Analysis of Genome Architecture during SCNT Reveals a Role of Cohesin in Impeding Minor ZGA.

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Coordination of zygotic genome activation entry and exit by H3K4me3 and H3K27me3 in porcine early embryos.

Histone modifications are critical epigenetic indicators of chromatin state associated with gene expression. Although the reprogramming patterns of H3K4me3 and H3K27me3 have been elucidated in mouse and human preimplantation embryos, the relationship between these marks and zygotic genome activation (ZGA) remains poorly understood. By ultra-low-input native chromatin immunoprecipitation and sequencing, we profiled global H3K4me3 and H3K27me3 in porcine oocytes and in vitro fertilized (IVF) embryos. We found that promoters of ZGA genes occupied sharp H3K4me3 peaks in oocytes, and these peaks became broader after fertilization, and reshaped into sharp again during ZGA. By simultaneous depletion of H3K4me3 demethylase KDM5B and KDM5C, we determined that broad H3K4me3 domain maintenance impaired ZGA gene expression, suggesting its function to prevent premature ZGA entry. By contrast, broad H3K27me3 domains underwent global removal upon fertilization, followed by a re-establishment for H3K4me3/H3K27me3 bivalency in morulae. We also found that bivalent marks were deposited at promoters of ZGA genes, and inhibiting this deposition was correlated with the activation of ZGA genes. It suggests that promoter bivalency contributes to ZGA exit in porcine embryos. Moreover, we demonstrated that aberrant reprogramming of H3K4me3 and H3K27me3 triggered ZGA dysregulation in somatic cell nuclear transfer (SCNT) embryos, whereas H3K27me3-mediated imprinting did not exist in porcine IVF and SCNT embryos. Our findings highlight two previously unknown epigenetic reprogramming modes coordinated with ZGA in porcine preimplantation embryos. Finally, the similarities observed between porcine and human histone modification dynamics suggest that the porcine embryo may also be a useful model for human embryo research.

OCT4 regulates WNT/ß-catenin signaling and prevents mesoendoderm differentiation by repressing EOMES in porcine pluripotent stem cells.

The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self-renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/ß-catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/ß-catenin signaling because the target gene of WNT/ß-catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/ß-catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription.

Nicotine exposure disrupts placental development via the Notch signaling pathway.

In brief: Normal gene expression during early embryonic development and in the placenta is crucial for a successful pregnancy. Nicotine can disrupt normal gene expression during development, leading to abnormal embryonic and placental development. Abstract: Nicotine is a common indoor air pollutant that is present in cigarette fumes. Due to its lipophilic nature, nicotine can rapidly transport through membrane barriers and spread throughout the body, which can lead to the development of diseases. However, the impact of nicotine exposure during early embryonic development on subsequent development remains elusive. In this study, we found that nicotine significantly elevated reactive oxygen species, DNA damage and cell apoptosis levels with the decrease of blastocyst formation during early embryonic development. More importantly, nicotine exposure during early embryonic development increased placental weight and disrupted placental structure. In molecular level, we also observed that nicotine exposure could specifically cause the hypermethylation of Phlda2 promoter (a maternally expressed imprinted gene associated with placental development) and reduce the mRNA expression of Phlda2. By RNA sequencing analysis, we demonstrated that nicotine exposure affected the gene expression and excessive activation of the Notch signaling pathway thereby affecting placental development. Blocking the Notch signaling pathway by DAPT treatment could recover abnormal placental weight and structure induced by nicotine exposure. Taken together, this study indicates that nicotine causes the declining quality of early embryos and leads to placental abnormalities related to over-activation of the Notch signaling pathway.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications.

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Preserving Porcine Genetics: A Simple and Effective Method for On-Site Cryopreservation of Ear Tissue Using Direct Cover Vitrification.

Cell cryopreservation is widely used for porcine genetic conservation; however, isolating and freezing primary cells in farms without adequate experimental equipment and environment poses a significant challenge. Therefore, it is necessary to establish a quick and simple method to freeze tissues on-site, which can be used for deriving primary fibroblasts when needed to achieve porcine genetic conservation. In this study, we explored a suitable approach for porcine ear tissue cryopreservation. The porcine ear tissues were cut into strips and frozen by direct cover vitrification (DCV) in the cryoprotectant solution with 15% EG, 15% DMSO and 0.1 M trehalose. Histological analysis and ultrastructural evaluation revealed that thawed tissues had normal tissue structure. More importantly, viable fibroblasts could be derived from these tissues frozen in liquid nitrogen for up to 6 months. Cells derived from thawed tissues did not show any cell apoptosis, had normal karyotypes and could be used for nuclear transfer. These results suggest that this quick and simple ear tissue cryopreservation method can be applied for porcine genetic conservation, especially in the face of a deadly emerging disease in pigs.

Lipidomic Characterization of Oocytes at Single-Cell Level Using Nanoflow Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Mass spectrometry (MS)-based lipidomic has become a powerful tool for studying lipids in biological systems. However, lipidome analysis at the single-cell level remains a challenge. Here, we report a highly sensitive lipidomic workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS)-MS. This approach enables the high-coverage identification of lipidome landscape at the single-oocyte level. By using the proposed method, comprehensive lipid changes in porcine oocytes during their maturation were revealed. The results provide valuable insights into the structural changes of lipid molecules during porcine oocyte maturation, highlighting the significance of sphingolipids and glycerophospholipids. This study offers a new approach to the single-cell lipidomic.

Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation.

In female meiosis, oocyte meiotic maturation is a form of asymmetric cell division, producing the first polar body and a large oocyte, in which the asymmetry of oocyte meiotic division depends on spindle migration and positioning, and cortical polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays an important role in asymmetric meiotic division during mouse oocyte maturation. Our initial study demonstrated that WDR62 mainly co-localized with chromosomes during mouse oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not affect germinal vesicle breakdown (GVBD) but compromised the first polar body extrusion (PBE) with the large polar bodies generated, which is coupled with a higher incidence of spindle abnormality and chromosome misalignment. Further analysis concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap formation, which should be responsible for large polar body extrusion. Moreover, WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but not expression level. In summary, our study defines WDR62 as an essential cytoskeletal regulator of spindle migration and asymmetric division during mouse oocyte meiotic maturation.

Single-cell RNA-seq reveals mRNAs and lncRNAs important for oocytes in vitro matured in pigs.

The faithful execution of molecular programme underlying oocyte maturation and meiosis is vital to generate competent haploid gametes for efficient mammalian reproduction. However, the organization and principle of molecular circuits and modules for oocyte meiosis remain obscure. Here, we employed the recently developed single-cell RNA-seq technique to profile the transcriptomes of germinal vesicle (GV) and metaphase II (MII) oocytes, aiming to discover the dynamic changes of mRNAs and long non-coding RNAs (lncRNAs) during oocyte in vitro meiotic maturation. During the transition from GV to MII, total number of detected RNAs (mRNAs and lncRNAs) in oocytes decreased. Moreover, 1,807 (602 up- and 1,205 down-regulated) mRNAs and 313 (177 up- and 136 down-regulated) lncRNAs were significantly differentially expressed (DE), i.e., more mRNAs down-regulated, but more lncRNAs up-regulated. During maturation of pig oocytes, mitochondrial mRNAs were actively transcribed, eight of which (ND6, ND5, CYTB, ND1, ND2, COX1, COX2 and COX3) were significantly up-regulated. Both DE mRNAs and targets of DE lncRNAs were enriched in multiple biological and signal pathways potentially associated with oocyte meiosis. Highly abundantly expressed mRNAs (including DNMT1, UHRF2, PCNA, ARMC1, BTG4, ASNS and SEP11) and lncRNAs were also discovered. Weighted gene co-expression network analysis (WGCNA) revealed 20 hub mRNAs in three modules to be important for oocyte meiosis and maturation. Taken together, our findings provide insights and resources for further functional investigation of mRNAs/lncRNAs in in vitro meiotic maturation of pig oocytes.

Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes.

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore-microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single-cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F-actin and spindle), and decreased DNA 5-methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC-hydroxylase ten-11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1-cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells.

Melatonin protects against defects induced by malathion during porcine oocyte maturation.

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

sRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs.

MOTIVATION: Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. RESULTS: The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531 306 and 2 941 669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. AVAILABILITY AND IMPLEMENTATION: sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Induction of autophagy promotes porcine parthenogenetic embryo development under low oxygen conditions.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.

The toxic effects of Fluorene-9-bisphenol on porcine oocyte in vitro maturation.

Fluorene-9-bisphenol (9,9-bis(4-hydroxyphenyl)-fluorene [BHPF]) is a bisphenol A (BPA) substitute used in the production of "BPA-free" plastics, now has been identified is harmful to living organisms. Our previous study showed that BHPF impaired mouse denuded oocyte in vitro maturation. However, there is a question that whether BHPF is still able to affect oocyte maturation in the presence of dense cumulus cells. In the present study, we checked the toxic effects of BHPF on porcine oocyte maturation which is derived from COCs in vitro culture. Our results showed that BHPF (50 µM) inhibited the expansion of cumulus cells, led to a significant decrease in polar body extrusion (PBE). Importantly, BHPF resulted in abnormal spindle assembly, ATP level decrease, reactive oxygen species (ROS) accumulation and early apoptosis in porcine oocytes, which are all negative to oocyte maturation. Furthermore, BHPF also declined porcine oocyte quality by disturbing the cortical granules (CGs) distribution. In conclusion, our study showed that BHPF still inhibited oocyte maturation even in the presence of cumulus cells leading to abnormal spindle assembly, ATP decrease, increased ROS level, early apoptosis, and disturbed CGs distribution in porcine oocytes, and also indicates that BHPF has a wide range toxic effects on oocyte in different species.

Reelin regulates male mouse reproductive capacity via the sertoli cells.

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells.

Nucleoporin35 is a novel microtubule associated protein functioning in oocyte meiotic spindle architecture.

Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment.