Publications by authors named "Paul W Dyce"

43 Publications

YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway.

Histochem Cell Biol 2021 Sep 29. Epub 2021 Sep 29.

College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, 266109, China.

Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of SDSCs are a hot topic. In this paper, we explore the link between the transcriptional regulator yes-associated protein (YAP) and the fate of porcine SDSCs (pSDSCs). We found that lysophosphatidylcholine (LPC) activates YAP, promotes pSDSCs pluripotency, and counteracts transdifferentiation of pSDSCs into porcine primordial germ cell-like cells (pPGCLCs). YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Oct4 and Sox2. The overexpression of YAP prevented the differentiation of pSDSCs, and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/β-catenin signaling pathway. When an activator of the Wnt/β-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP, the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939, an inhibitor of the Wnt/β-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Taken together, our results suggested that YAP and the Wnt/β-catenin signaling pathway interact to regulate the fate of pSDSCs.
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http://dx.doi.org/10.1007/s00418-021-02034-4DOI Listing
September 2021

Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice.

PLoS Biol 2020 12 22;18(12):e3001025. Epub 2020 Dec 22.

College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, China.

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.
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http://dx.doi.org/10.1371/journal.pbio.3001025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787681PMC
December 2020

Dissecting the initiation of female meiosis in the mouse at single-cell resolution.

Cell Mol Life Sci 2021 Jan 4;78(2):695-713. Epub 2020 May 4.

College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China.

Meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single-cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By analysing cluster-specific gene expression profiles, we found four cell clusters correspond to different cell stages en route to meiosis and characterized their detailed transcriptome dynamics. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving female meiosis initiation.
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http://dx.doi.org/10.1007/s00018-020-03533-8DOI Listing
January 2021

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Comp Biochem Physiol A Mol Integr Physiol 2020 05 17;243:110675. Epub 2020 Feb 17.

School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, United States. Electronic address:

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin β (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.
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http://dx.doi.org/10.1016/j.cbpa.2020.110675DOI Listing
May 2020

Whole-transcriptome analysis of the toxic effects of zearalenone exposure on ceRNA networks in porcine granulosa cells.

Environ Pollut 2020 Jun 24;261:114007. Epub 2020 Jan 24.

Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China. Electronic address:

Zearalenone (ZEA), an estrogen-like mycotoxin, is commonly detected in animal feeds including improperly stored grains. It has been well demonstrated that ovarian granulosa cells (GCs) perform vital roles during follicular development, however, the competing endogenous RNA (ceRNA) network in GCs after ZEA exposure remains to be well described. Here, for the first time, we adopted whole-transcriptome sequence technology to explore the molecular mechanism of ZEA toxicology on porcine GCs. The results provide evidence that the cell cycle of porcine GCs is arrested in the G2/M phase after exposure to ZEA. Furthermore, bioinformation analysis found that cell cycle arrest related genes were perturbed, including CDK1, CCNB1, CDC25A, and CDC25C, which was consistent with the results of RT-qPCR, immunofluorescence, and Western Blotting. Based on the whole-transcriptome sequence data, by constructing ceRNA networks related to cell cycle arrest, we observed that ZEA exposure arrested cell cycle progression at the G2/M phase in porcine GCs, and non-coding RNAs (ncRNAs) played an important role in this process via regulating the expressions of cell cycle arrest related genes. Taken together, our data here provides strong data to support that the toxicological mechanism regarding the widely distributed toxicant ZEA acts through ceRNA networks in porcine granulosa cells.
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http://dx.doi.org/10.1016/j.envpol.2020.114007DOI Listing
June 2020

All-trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes.

Mol Reprod Dev 2019 12 22;86(12):1865-1873. Epub 2019 Sep 22.

Department of Animal Sciences, College of Agriculture, Auburn University, Auburn, Alabama.

In developing follicles, cellular coupling within cumulus-oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All-trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.
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http://dx.doi.org/10.1002/mrd.23274DOI Listing
December 2019

Evaluation of age, weaning weight, body condition score, and reproductive tract score in pre-selected beef heifers relative to reproductive potential.

J Anim Sci Biotechnol 2019 26;10:18. Epub 2019 Feb 26.

1Department of Animal Sciences, Auburn University, 559 Devall Dr, Auburn, AL 36839 USA.

Background: Artificial insemination is a preferred breeding method for beef heifers as it advances the genetic background, produces a predictive and profitable calving season, and extends the heifer's reproductive life span. As reproductive efficiency in heifers is key for the success of beef cattle production systems, following artificial insemination, heifers are exposed to a bull for the remainder of the breeding season. Altogether, up to 95% of heifers might become pregnant in their first breeding season. Heifers that do not become pregnant at the end of the breeding season represent an irreparable economical loss. Additionally, heifers conceiving late in the breeding season to natural service, although acceptable, poses serious losses to producers. To minimize losses due to reproductive failure, different phenotypic parameters can be assessed and utilized as selection tools. Here, we tested the hypothesis that in a group of pre-selected heifers, records of weaning weight, age at weaning, age at artificial insemination, and age of dam differ among heifers of varied reproductive outcomes during the first breeding season.

Results: None of the parameters tested presented predictive ability to discriminate the heifers based on the response variable ('pregnant to artificial insemination', 'pregnant to natural service', 'not pregnant'). Heifers categorized with body condition score = 6 and reproductive tract score ≥ 4 had the greatest proportion of pregnancy to artificial insemination (49% and 44%, respectively). Furthermore, it was notable that heifers presenting body condition score = 6 and reproductive tract score = 5 presented the greatest pregnancy rate at end of the breeding season (89%). Heifers younger than 368 d at the start of the breeding season did not become pregnant to artificial insemination. Those young heifers had 12.5% chance to become pregnant in their first breeding season, compared to 87.5% if the heifers were older than 368 days.

Conclusion: Our results suggest that beef heifers with body condition score = 6 and reproductive tract score ≥ 4 are more likely to become pregnant to artificial insemination. Careful assessment should be undertaken when developing replacement heifers that will not reach 12 months of age by the beginning of the breeding season.
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http://dx.doi.org/10.1186/s40104-019-0329-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390375PMC
February 2019

RA promotes proliferation of primordial germ cell-like cells differentiated from porcine skin-derived stem cells.

J Cell Physiol 2019 08 11;234(10):18214-18229. Epub 2019 Mar 11.

College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China.

Previous studies have shown that primordial germ cell-like cells (PGCLCs) can be obtained from human, porcine and mouse skin-derived stem cells (SDSCs). In this paper, we found retinoic acid (RA), the active derivative of vitamin A, accelerated the growth of porcine primordial germ cells (pPGCs) and porcine PGCLCs (pPGCLCs) which were derived from porcine SDSCs (pSDSCs). Moreover, flow cytometry results revealed that the proliferation promoting effect of RA was attenuated by U0126, a specific inhibitor of extracellular signal-regulated kinase (ERK). Western blot analysis showed the protein level of ERK, phosphorylated ERK, cyclin D1 (CCND1), and cyclin-dependent kinase 2 (CDK2) increased after stimulation with RA, and this effect could also be abolished by U0126. Our data revealed that ablation of ERK expression by U0126 should significantly decrease proliferation of pPGCLCS. This reduction was because CCND1 and CDK2 proteins level decrease and subsequently the pPGCLCs were arrested in the G0/G1 phase. In addition, we also confirmed RA indeed promoted the proliferation of pPGCs isolated from porcine fetal genital ridges in vitro. Furthermore, our data indicated that DNA methylation pattern were changed in pPGCLCs and this pattern were more similar to pPGCs.
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http://dx.doi.org/10.1002/jcp.28454DOI Listing
August 2019

Establishment and depletion of the ovarian reserve: physiology and impact of environmental chemicals.

Cell Mol Life Sci 2019 May 27;76(9):1729-1746. Epub 2019 Feb 27.

College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, 266109, China.

The reproductive life span in women starts at puberty and ends at menopause, following the exhaustion of the follicle stockpile termed the ovarian reserve. Increasing data from experimental animal models and epidemiological studies indicate that exposure to a number of ubiquitously distributed reproductively toxic environmental chemicals (RTECs) can contribute to earlier menopause and even premature ovarian failure. However, the causative relationship between environmental chemical exposure and earlier menopause in women remains poorly understood. The present work, is an attempt to review the current evidence regarding the effects of RTECs on the main ovarian activities in mammals, focusing on how such compounds can affect the ovarian reserve at any stages of ovarian development. We found that in rodents, strong evidence exists that in utero, neonatal, prepubescent and even adult exposure to RTECs leads to impaired functioning of the ovary and a shortening of the reproductive lifespan. Regarding human, data from cross-sectional surveys suggest that human exposure to certain environmental chemicals can compromise a woman's reproductive health and in some cases, correlate with earlier menopause. In conclusion, evidences exist that exposure to RTECs can compromise a woman's reproductive health. However, human exposures may date back to the developmental stage, while the adverse effects are usually diagnosed decades later, thus making it difficult to determine the association between RTECs exposure and human reproductive health. Therefore, epidemiological surveys and more experimental investigation on humans, or alternatively primates, are needed to determine the direct and indirect effects caused by RTECs exposure on the ovary function, and to characterize their action mechanisms.
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http://dx.doi.org/10.1007/s00018-019-03028-1DOI Listing
May 2019

Ochratoxin A Exposure Impairs Porcine Granulosa Cell Growth via the PI3K/AKT Signaling Pathway.

J Agric Food Chem 2019 Mar 25;67(9):2679-2690. Epub 2019 Feb 25.

College of Life Sciences, Institute of Reproductive Sciences , Qingdao Agricultural University , Qingdao 266109 , China.

The mycotoxin ochratoxin A (OTA), a naturally occurring food contaminant, has a toxic effect on the growth and development of follicles in pigs. However, little is known regarding the specific toxic effects of OTA exposure on oocytes and granulosa cells (GCs). In this study, we cultured porcine ovarian GCs and exposed them to OTA in vitro in order to explore the mechanism causing the negative effects. Initially, it was found that OTA exposure inhibited cell viability in a time and dose dependent manner. We also showed that OTA exposure increased oxidative stress, decreased proliferation ratio, and increased apoptosis ratio in GCs. We revealed an important role for the PI3K/AKT signal pathway in GC proliferation and apoptosis by RNA-seq analysis. The results not only showed that OTA treatment significantly affected the expression of genes within the PI3K/AKT pathway but also demonstrated a concrete relationship between the PI3K/AKT pathway and GC cell proliferation and apoptosis. In conclusion, the results demonstrated that OTA exposure impaired porcine GC growth via the PI3K/AKT signaling pathway.
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http://dx.doi.org/10.1021/acs.jafc.8b06361DOI Listing
March 2019

Plasma metabolomic profiles differ at the time of artificial insemination based on pregnancy outcome, in Bos taurus beef heifers.

Sci Rep 2018 09 4;8(1):13196. Epub 2018 Sep 4.

Department of Animal Sciences, College of Agriculture, Auburn University, Auburn, AL, 36849, USA.

Infertility remains the most prevalent reason for cattle being removed from production environments. We utilized metabolomic profiling to identify metabolites in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Prior to AI, phenotypic parameters including body condition, weight, and reproductive organ measurements were collected. These were determined not effective at differentiating between fertile and infertile heifers. Analysis of the resulting metabolomic profiles revealed 15 metabolites at significantly different levels (T-test P ≤ 0.05), with seven metabolites having a greater than 2-fold difference (T-test P ≤ 0.05, fold change ≥2, ROC-AUC ≥ 0.80) between infertile and fertile heifers. We further characterized the utility of using the levels of these metabolites in the blood plasma to discriminate between fertile and infertile heifers. Finally, we investigated the potential role inflammation may play by comparing the expression of inflammatory cytokines in the white blood cells of infertile heifers to that of fertile heifers. We found significantly higher expression in infertile heifers of the proinflammatory markers tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), and the C-X-C motif chemokine 5 (CXCL5). Our work offers potentially valuable information regarding the diagnosis of fertility problems in heifers undergoing AI.
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http://dx.doi.org/10.1038/s41598-018-31605-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123494PMC
September 2018

Exposure to Zinc oxide nanoparticles during pregnancy induces oocyte DNA damage and affects ovarian reserve of mouse offspring.

Aging (Albany NY) 2018 Aug;10(8):2170-2189

College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109, China.

Zinc oxide nanoparticles (nZnO) have been shown to have higher toxic effects likely due to their ion-shedding ability and low solubility under neutral conditions. In order to investigate whether exposure to nZnO during embryonic development affects ovary development, 12.5 day post coitum (dpc) fetal mouse ovaries were cultured in the presence of nZnO for 6 days. We found that the nanoparticles (NPs) accumulated within the oocyte cytoplasm in a dose dependent manner, caused DNA damage and apoptosis, and result in a significant decrease in oocyte numbers. No such effects were observed when the ovaries were incubated in the presence of ZnSO or bulk ZnO as controls. In addition, we injected intravenously 16 mg/kg body weight nZnO in 12.5 dpc pregnant mice on two consecutive days and analyzed the ovaries of fetuses or offspring at three critical periods of oogenesis: 17.5 dpc, 3 days post-partum (dpp) and 21 dpp. Evidence of increased DNA damage in pachytene oocytes in fetal ovaries and impaired primordial follicle assembly and folliculogenesis dynamics in the ovaries of the offspring were found. Our results indicate that certain types of NPs affect pre- and post-natal oogenesis and .
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http://dx.doi.org/10.18632/aging.101539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128443PMC
August 2018

The impact of epidermal growth factor supernatant on pig performance and ileal microbiota.

Transl Anim Sci 2018 Apr 18;2(2):184-194. Epub 2018 May 18.

Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada.

Weaning of pigs can lead to low-feed intake resulting in a lag in growth performance, reduced gut health, and diarrheal diseases. Epidermal growth factor (), the most abundant growth factor in milk, increased weaned pig BW gain and feed efficiency in our previous work. It is believed that intestinal microbiota plays an important role in gut health and pig growth, but limited data are available on the impact of feed additives, such as EGF, on the microbial communities of the intestines. The objective of the study was to investigate if the positive influence of EGF supplementation on weight gain and gut health was related to differences in intestinal microbiota. To examine the efficacy of EGF, a 21-d animal trial was performed using 72 pigs (two equal blocks of 36 pigs with three barrows and three gilts/pen). Pigs were assigned to one of two dietary treatments at weaning (20 ± 2 d of age; = 6 pens/treatment) balancing across treatment for litter, gender, and initial BW. Recombinant yeast supernatant containing EGF at 120 μg/kg BW/d and without EGF (control) was added to the feed for 21 d, followed by a common diet for 7 d. Pig performance was measured weekly and ileal digesta was collected at day 21 from six pigs/treatment for microbiome analysis. Pigs fed diets containing EGF fermentation supernatant had greater ( = 0.01) daily gain in week 3 and overall resulting in heavier ( = 0.029) BW at day 28, which was consistent to our previous finding. No difference in alpha-diversity (Chao1, Shanon, and Simpson indices) and beta-diversity (weighted and unweighted UniFrac distances) of ileal digesta microbiota between EGF supplemented and control pigs were observed. The relative abundances of bacterial taxa did not differ among treatment groups at the phylum level. The relative abundances of (0.0 vs. 0.9%), (0.003 vs. 0.26%), and (0.0 vs. 0.05%) genera, and Rumminococcaceae family (0.001 vs. 0.08%) were decreased ( < 0.05) in EGF group compared to control and were negatively correlated ( < 0.05, r > 0.60) with growth performance. Pathways related to detoxification and carbohydrate metabolism were differentially represented in the luminal bacterial populations. The improved growth of pigs supplemented with EGF supernatant produced by may be related to changes in functional capacity of the gut microbial populations. However, the impact on mucosa-associated or large intestinal communities is still unknown.
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http://dx.doi.org/10.1093/tas/txy019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200883PMC
April 2018

Connexin 43 coupling in bovine cumulus cells, during the follicular growth phase, and its relationship to in vitro embryo outcomes.

Mol Reprod Dev 2018 07 23;85(7):579-589. Epub 2018 May 23.

Department of Animal Sciences, College of Agriculture, Auburn University, Auburn, Alabama.

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two-cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.
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http://dx.doi.org/10.1002/mrd.22993DOI Listing
July 2018

Isolation of bacteria from fermented food and grass carp intestine and their efficiencies in improving nutrient value of soybean meal in solid state fermentation.

J Anim Sci Biotechnol 2018 5;9:29. Epub 2018 Apr 5.

1Department of Animal BioSciences, University of Guelph, Guelph, Canada.

Background: Soybean meal is an excellent and cost-effective protein source; however, its usage is limited in the piglet due to the presence of anti-nutritional factors and the antigens glycinin and β-conglycinin. The objective of the current study was to screen and select for bacteria that can be efficiently adopted to ferment soybean meal in order to solve this problem.

Results: Bacteria were isolated from fermented soy foods and the grass carp intestine, and strains selected for high protease, cellulase and amylase activities. The isolated bacteria were characterized as respectively. Fermentation with food-derived Isolate-2 and fish-derived F-9 increased crude protein content by 5.32% and 8.27%, respectively; improved the amino acid profile by increasing certain essential amino acids, broke down larger soy protein to 35 kDa and under, eliminated antigenicity against glycinin and β-conglycinin, and removed raffinose and stachyose in the soybean meal following a 24-h fermentation.

Conclusions: Our results suggest these two bacteria can efficiently solid state ferment soybean meal and ultimately produce a more utilizable food source for growing healthy piglets.
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http://dx.doi.org/10.1186/s40104-018-0245-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885361PMC
April 2018

Retinoic acid enhances germ cell differentiation of mouse skin-derived stem cells.

J Ovarian Res 2018 Mar 1;11(1):19. Epub 2018 Mar 1.

Department of Physiology and Pharmacology, The University of Western Ontario and Children's Health Research Institute, 800 Commissioners Road East, London, ON, N6C 2V5, Canada.

Background: Retinoic acid (RA) signaling has been identified as a key driver in male and female gamete development. The presence of RA is a critical step in the initiation of meiosis and is required for the production of competent oocytes from primordial germ cells. Meiosis has been identified as a difficult biological process to recapitulate in vitro, when differentiating stem cells to germ cells. We have previously shown that primordial germ cell-like cells, and more advanced oocyte-like cells (OLCs), can be formed by differentiating mouse skin-derived stem cells. However, the OLCs remain unable to function due to what appears to be failure of meiotic initiation. The aim of this study was to determine the effect of RA treatment, during stem cell differentiation to germ cells, particularly on the initiation of meiosis.

Results: Using qPCR we found significant increases in the meiosis markers Stra8 and Sycp3 and a significant reduction in the meiosis inhibitor Nanos2, in the differentiating populations. Furthermore, OLCs from the RA treated group, expressed significantly more of the meiosis regulatory gene Marf1 and the oocyte marker Oct4. At the protein level RA treatment was found to increase the expression of the gap junction protein CX43 and the pluripotency marker OCT4. Moreover, the expression of SYCP3 was significantly upregulated and the localization pattern better matched that of control fetal ovarian cells. RA treatment also improved the structural integrity of the OLCs produced by initiating the expression of all three zona pellucida transcripts (Zp1-3) and improving ZP3 expression levels and localization. Finally, the addition of RA during differentiation led to an almost two-fold increase in the number of OLCs recovered and increased their in vitro growth.

Conclusion: RA is a key driver in the formation of functioning gametes and its addition during stem cell to germ cell differentiation improves OLCs entry into meiosis.
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http://dx.doi.org/10.1186/s13048-018-0390-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831580PMC
March 2018

Transcriptome profiles in peripheral white blood cells at the time of artificial insemination discriminate beef heifers with different fertility potential.

BMC Genomics 2018 02 9;19(1):129. Epub 2018 Feb 9.

Department of Animal Sciences, Auburn University, 559 Devall Dr, Auburn, AL, 36839, USA.

Background: Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant.

Results: We generated RNA-sequencing data from 23 heifers from two locations (A: six AI-pregnant and five NB-pregnant; and B: six AI-pregnant and six non-pregnant). After filtering out lowly expressed genes, we quantified transcript abundance for 12,538 genes. The comparison of gene expression levels between AI-pregnant and NB-pregnant heifers yielded 18 differentially expressed genes (DEGs) (ADAM20, ALDH5A1, ANG, BOLA-DQB, DMBT1, FCER1A, GSTM3, KIR3DL1, LOC107131247, LOC618633, LYZ, MNS1, P2RY12, PPP1R1B, SIGLEC14, TPPP, TTLL1, UGT8, eFDR≤0.02). The comparison of gene expression levels between AI-pregnant and non-pregnant heifers yielded six DEGs (ALAS2, CNKSR3, LOC522763, SAXO2, TAC3, TFF2, eFDR≤0.05). We calculated the ratio of expression levels between all gene pairs and assessed their potential to classify samples according to experimental groups. Considering all samples, relative expression from two gene pairs correctly classified 10 out of 12 AI-pregnant heifers (P = 0.0028) separately from the other 11 heifers (NB-pregnant, or non-pregnant).

Conclusion: The transcriptome profile in PWBC, at the time of AI, is associated with the fertility potential of beef heifers. Transcript levels of specific genes may be further explored as potential classifiers, and thus selection tools, of heifer fertility.
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http://dx.doi.org/10.1186/s12864-018-4505-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807776PMC
February 2018

Epigenetic regulation during the differentiation of stem cells to germ cells.

Oncotarget 2017 Aug 12;8(34):57836-57844. Epub 2017 Jun 12.

College of Animal Science and Technology, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109, China.

Gametogenesis is an essential process to ensure the transfer of genetic information from one generation to the next. It also provides a mechanism by which genetic evolution can take place. Although the genome of primordial germ cells (PGCs) is exactly the same with somatic cells within an organism, there are significant differences between their developments. For example, PGCs eventually undergo meiosis to become functional haploid gametes, and prior to that they undergo epigenetic imprinting which greatly alter their genetic regulation. Epigenetic imprinting of PGCs involves the erasure of DNA methylation and the reestablishment of them during sperm and oocyte formation. These processes are necessary and important during gametogenesis. Also, histone modification and X-chromosome inactivation have important roles during germ cell development. Recently, several studies have reported that functional sperm or oocytes can be derived from stem cells or . To produce functional germ cells, induction of germ cells from stem cells must recapitulate these processes similar to endogenous germ cells, such as epigenetic modifications. This review focuses on the epigenetic regulation during the process of germ cell development and discusses their importance during the differentiation from stem cells to germ cells.
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http://dx.doi.org/10.18632/oncotarget.18444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593687PMC
August 2017

Oocyte-like cells induced from CD34-positive mouse hair follicle stem cells in vitro.

J Genet Genomics 2017 08 12;44(8):405-407. Epub 2017 Aug 12.

Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA.

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http://dx.doi.org/10.1016/j.jgg.2017.08.001DOI Listing
August 2017

Complete in vitro oogenesis: retrospects and prospects.

Cell Death Differ 2017 11 25;24(11):1845-1852. Epub 2017 Aug 25.

Institute of Reproductive Sciences, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.

Precise control of mammalian oogenesis has been a traditional focus of reproductive and developmental biology research. Recently, new reports have introduced the possibility of obtaining functional gametes derived in vitro from stem cells. The potential to produce functional gametes from stem cells has exciting applications for regenerative medicine though still remains challenging. In mammalian females ovulation and fertilization is a privilege reserved for a small number of oocytes. In reality the vast majority of oocytes formed from primordial germ cells (PGCs) will undergo apoptosis, or other forms of cell death. Removal occurs during germ cell cyst breakdown and the establishment of the primordial follicle (PF) pool, during the long dormancy at the PF stage, or through follicular atresia prior to reaching the ovulatory stage. A way to solve this limitation could be to produce large numbers of oocytes, in vitro, from stem cells. However, to recapitulate mammalian oogenesis and produce fertilizable oocytes in vitro is a complex process involving several different cell types, precise follicular cell-oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. The present article offers a critical overview of these results as well as other previous work performed mainly in mouse attempting to reproduce oogenesis completely in vitro and considers some perspectives for the potential to adapt the methods to produce functional human oocytes.
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http://dx.doi.org/10.1038/cdd.2017.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635224PMC
November 2017

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

Cell Death Dis 2017 08 3;8(8):e2966. Epub 2017 Aug 3.

College of Animal Science and Technology, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109, China.

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.
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http://dx.doi.org/10.1038/cddis.2017.350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596541PMC
August 2017

The role of germ cell loss during primordial follicle assembly: a review of current advances.

Int J Biol Sci 2017 11;13(4):449-457. Epub 2017 Mar 11.

College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling Shaanxi 712100, China.

In most female mammals, early germline development begins with the appearance of primordial germ cells (PGCs), and develops to form mature oocytes following several vital processes. It remains well accepted that significant germ cell apoptosis and oocyte loss takes place around the time of birth. The transition of the ovarian environment from fetal to neonatal, coincides with the loss of germ cells and the timing of follicle formation. All told it is common to lose approximately two thirds of germ cells during this transition period. The current consensus is that germ cell loss can be attributed, at least in part, to programmed cell death (PCD). Recently, autophagy has been implicated as playing a part in germ cell loss during the time of parturition. In this review, we discuss the major opinions and mechanisms of mammalian ovarian PCD during the process of germ cell loss. We also pay close attention to the function of autophagy in germ cell loss, and speculate that autophagy may also serve as a critical and necessary process during the establishment of primordial follicle pool.
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http://dx.doi.org/10.7150/ijbs.18836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436565PMC
February 2018

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

Nanotoxicology 2017 May 6;11(4):465-474. Epub 2017 Apr 6.

a Institute of Reproductive Sciences, Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University , Qingdao , Shandong , China.

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.
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http://dx.doi.org/10.1080/17435390.2017.1310947DOI Listing
May 2017

The epigenetic modifications and the anterior to posterior characterization of meiotic entry during mouse oogenesis.

Histochem Cell Biol 2017 Jul 24;148(1):61-72. Epub 2017 Feb 24.

College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China.

The meiotic initiation of mammalian oogonia is a critical step during the development of primordial germ cells (PGCs) to mature oocytes. In this study, a systematic investigation of epigenetic modifications and DAZL gene expression during oogonia meiotic entry were performed. We found that the expression of DAZL was epigenetically regulated by DNA methylation of CpG islands within its promoter region. During meiotic entry, a continuously increasing level of 5hmC, a stable epigenetic marker usually associated with the activation of gene expression, was observed from 11.5 to 16.5 dpc (days post coitum). Meanwhile trimethylation of lysine 27 on histone3 (H3K27me3), usually associated with repression of gene expression, had a sustainable increase from 12.5 to 16.5 dpc. Finally, by equally dividing the ovaries into three regions representing the anterior, the middle, and the posterior of the ovary and performing immunofluorescence and qRT-PCR on the individual regions, we provided further evidences that the meiotic entry and progression of female germ cells is in an anterior to posterior pattern.
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http://dx.doi.org/10.1007/s00418-017-1545-9DOI Listing
July 2017

Skin-derived stem cells as a source of primordial germ cell- and oocyte-like cells.

Cell Death Dis 2016 11 10;7(11):e2471. Epub 2016 Nov 10.

Institute of Reproductive Sciences, Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China.

The skin is a unique organ that contains a variety of stem cells for the maintenance of skin homeostasis and the repair of skin tissues following injury and disease. Skin-derived stem cells (SDSCs) constitute a heterogeneous population of stem cells generated in vitro from dermis, which can be cultured as spherical aggregates of cells in suspension culture. Under certain in vitro or in vivo conditions, SDSCs show multipotency and can generate a variety of neural, mesodermal, and endodermal cell types such as neurons, glia, fibroblasts, adipocytes, muscle cells, chondroblasts, osteoblats, and islet β-cell-like cells. SDSCs are likely derived from multipotent stem cells located in the hair follicles that are, in turn, derived from embryonic migratory neural crest or mesoderm cells. During the past decade, a wave of reports have shown that germ cells can be generated from various types of stem cells. It has been shown that SDSCs are able to produce primordial germ cell-like cells in vitro, and even oocyte-like cells (OLCs). Whether these germ cell-like cells (GCLCs) can give rise to viable progeny remains, however, unknown. In this review, we will discuss the origin and characteristics of SDSCs from which the GCLC are derived, the possible mechanisms of this differentiation process, and finally the prospective biomedical applications of the SDSC-derived GCLCs.
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http://dx.doi.org/10.1038/cddis.2016.366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260893PMC
November 2016

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

Arch Toxicol 2017 Mar 12;91(3):1279-1292. Epub 2016 Jul 12.

Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, 266109, China.

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, 17β-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.
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http://dx.doi.org/10.1007/s00204-016-1790-zDOI Listing
March 2017

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

PLoS One 2016 4;11(2):e0148350. Epub 2016 Feb 4.

College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148350PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741416PMC
August 2016

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca).

PLoS One 2015 16;10(9):e0137712. Epub 2015 Sep 16.

Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, Shandong, 266109, China; Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong, 266109, China.

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137712PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574107PMC
May 2016

Connexin43 is required for the maintenance of multipotency in skin-derived stem cells.

Stem Cells Dev 2014 Jul 15;23(14):1636-46. Epub 2014 May 15.

1 Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario , London, Ontario, Canada .

Expression of the gap junction protein, connexin43 (Cx43), begins early during embryogenesis and is maintained in many different cell types. Several stem cell populations have been shown to express Cx43 and to form functional gap junctions. While it is clear that Cx43 is critical to the function of many organs, whether the same is true for stem cells has not been clearly demonstrated. Recently, stem cells isolated from newborn mouse skin were shown to form oocyte-like cells (OLCs) in vitro, hence the present study focussed on the role Cx43 plays in the proliferation and differentiation of these cells. The stem cells express Cx43 and those from knockout mice (Cx43 KO) exhibited significantly reduced cell-cell coupling. Loss of Cx43 reduced the rate of cellular migration [Cx43 KO, 1.57±0.65 radial cell units (RCU); wildtype (WT), 5.57±0.37 RCU] but increased the proliferation rate of the stem cells (Cx43 KO, 29.40%±2.02%; WT, 12.76%±1.50%). The expression of the pluripotency markers OCT4 and Nanog were found to be reduced in the Cx43 KO population, suggesting an inhibition of differentiation potential. To test the differentiation ability, the stem cells were induced to form neuronal cell types in vitro. While both the WT and KO cells were able to form GFAP-positive astrocytic cells, only WT stem cells were able to form βIII tubulin-positive neurons. Similarly, the ability of the stem cells to form OLCs was ablated by the loss of Cx43. These data reveal a role for Cx43 in maintaining multipotency within the skin-derived stem cell population.
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http://dx.doi.org/10.1089/scd.2013.0459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086243PMC
July 2014

Phosphorylation of serine residues in the C-terminal cytoplasmic tail of connexin43 regulates proliferation of ovarian granulosa cells.

J Membr Biol 2012 Jun 24;245(5-6):291-301. Epub 2012 Jun 24.

Department of Physiology and Pharmacology, The University of Western Ontario and Children's Health Research Institute, London, ON N6C 2V5, Canada.

Connexin43 (Cx43) forms gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted as a result of impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279, and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells that were combined with wild-type oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development.
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http://dx.doi.org/10.1007/s00232-012-9450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578320PMC
June 2012
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