Paul W. Dyce

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.

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

Abstract 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.

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

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.

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.

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

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.

Complete in vitro oogenesis: retrospects and prospects

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.

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

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.

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

BackgroundInfertility 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.ResultsWe 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).ConclusionThe 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.

Epigenetic regulation during the differentiation of stem cells to germ cells

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 in vivo or in vitro. 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.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 in vivo or in vitro. 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.

The impact of epidermal growth factor supernatant on pig performance and ileal microbiota1

Abstract 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 (EGF), 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; n = 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 (P = 0.01) daily gain in week 3 and overall resulting in heavier (P = 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 Corynebacterium (0.0 vs. 0.9%), Blautia (0.003 vs. 0.26%), and Coprococcus (0.0 vs. 0.05%) genera, and Rumminococcaceae family (0.001 vs. 0.08%) were decreased (P < 0.05) in EGF group compared to control and were negatively correlated (P < 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 Pichia pastoris 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.