Isolation, culture and characterization of chicken primordial germ cells

2006 ◽  
Vol 3 (3) ◽  
pp. 183-188 ◽  
Author(s):  
Tang Xin-Yan ◽  
Zeng Wei-Dong ◽  
Mi Yu-Ling ◽  
Liu Hong-Yun ◽  
Zhang Cai-Qiao
Keyword(s):  
Germ Cells ◽  
Gradient Centrifugation ◽  
Periodic Acid ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Nuclear Antigen ◽  
Periodic Acid Schiff ◽  
Ficoll Density Gradient Centrifugation ◽  
Pas Staining ◽  
Proliferating Activity

AbstractPrimordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.

scholarly journals Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 43
Author(s):  
Qisheng Zuo ◽  
Jing Zhou ◽  
Man Wang ◽  
Yani Zhang ◽  
Guohong Chen ◽  
...  
Keyword(s):  
Germ Cells ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Periodic Acid ◽  
Primordial Germ Cells ◽  
Negative Regulator ◽  
Marker Genes ◽  
Periodic Acid Schiff

Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.

An ovomucin-like protein on the surface of migrating primordial germ cells of the chick and rat

Development ◽  
1996 ◽  
Vol 122 (3) ◽  
pp. 915-923
Author(s):  
W. Halfter ◽  
B. Schurer ◽  
H.M. Hasselhorn ◽  
B. Christ ◽  
E. Gimpel ◽  
...  
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Major Constituent ◽  
Vitelline Membrane ◽  
Relative Molecular Mass ◽  
Male Chick ◽  
Dissociated Cells ◽  
Pas Staining ◽  
Adhesive Quality

A mucin was discovered on the surface of migratory primordial germ cells (PGCs) from chick and rat embryos by means of two monoclonal antibodies. The protein was found to be identical or closely related to ovomucin, a 600 X 10(3) relative molecular mass glycoprotein, and a major constituent of the vitelline membrane of the avian yolk. Based on its resemblance to ovomucin it is referred to as ovomucin-like protein (OLP). The OLP was expressed on PGCs from E3 to E7 female, and from E3 to E12 male chick embryos as the PGCs migrate and colonize the gonadal ridges. After the PGCs have settled in the gonads, they no longer express OLP. In tissue cultures of dissociated cells from E6 gonads, OLP was present only on cells that were positive for PAS staining, the standard histological method to identify PGCs in the chick embryo. Since unfixed PGCs were recognized by the antibodies, at least part of the OLP is localized on the cell surface. The anti-OLP antibodies also stained PGCs in the gonads of the rat embryo, showing that the expression of this antigen on PGCs is phylogenetically conserved. Ovomucin isolated from vitelline membrane prevented adhesion of fibroblasts but not PGCs when used a as a substratum in vitro. The anti-adhesive quality of the mucin resides in the sialic acid residues of the carbohydrate side chains. We propose that OLP has a similar anti-adhesive quality as the ovomucin from vitelline membrane, and that this anti-adhesive property is important to prevent precocious adhesion of migrating PGCs to blood vessel walls and to connective tissue in the mesentery as they migrate toward the gonadal ridges.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals The Effect of Long-Term Use of an Eyewash Solution on the Ocular Surface Mucin Layer

2019 ◽  
Vol 20 (20) ◽  
pp. 5078 ◽  
Author(s):  
Hiroyuki Yazu ◽  
Naoyuki Kozuki ◽  
Murat Dogru ◽  
Ayako Shibasaki ◽  
Hiroshi Fujishima
Keyword(s):  
Ocular Surface ◽  
Periodic Acid ◽  
Tear Film ◽  
Periodic Acid Schiff ◽  
Dry Eyes ◽  
Staining Score ◽  
Related Quality ◽  
Pas Staining ◽  
Mucin Layer ◽  
A Prospective Study

The use of eyewash solutions in Japan, especially in patients with allergic conjunctivitis and contact lens wearers, has been increasing. Our aim was to investigate how the use of preservative-free eyewash solution in healthy eyes for one month affects corneal safety and ocular surface mucin. We analyzed 42 eyes of 21 individuals (17 males, four females; mean age: 36.1 ± 7.4 years) without ocular allergies, dry eyes, or other ocular diseases through a prospective study. Eyes were randomized to a wash group (group one) and a nonwash follow up group (group two). We evaluated the dry eye-related quality-of-life score (DEQS), tear film breakup time (TBUT), fluorescein staining score, mRNA expression of MUC5AC and MUC16, MUC16 immunohistochemistry, and MUC5AC periodic acid Schiff (PAS) staining. There was a significant decrease in DEQS scores after one month of eyewash use (p < 0.05). There were no significant differences in other evaluation items that were analyzed (all p > 0.05). Furthermore, no significant differences were observed between group one and group two in all endpoints (all p > 0.05). The results suggest that one month use of a nonpreserved eyewash solution has no detrimental effects on the tear film and the ocular surface mucins.

Renal vascular and tubulointerstitial inflammation and proliferation in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension

2007 ◽  
Vol 292 (6) ◽  
pp. F1858-F1866 ◽  
Author(s):  
Miguel L. Graciano ◽  
Cynthia R. Mouton ◽  
Matthew E. Patterson ◽  
Dale M. Seth ◽  
John J. Mullins ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Morphological Changes ◽  
Periodic Acid ◽  
Cell Number ◽  
Malignant Hypertension ◽  
Nuclear Antigen ◽  
Ang Ii ◽  
Transgenic Rats ◽  
Periodic Acid Schiff ◽  
Proliferating Cell

Transgenic rats with inducible ANG II-dependent malignant hypertension [TGR(Cyp1a1Ren2)] were generated by inserting the mouse Ren2 renin gene into the genome of the rat. The present study was performed to assess renal morphological changes occurring during the development of ANG II-dependent malignant hypertension in these rats. Male Cyp1a1-Ren2 rats ( n = 10) were fed normal rat food containing indole-3-carbinol (I3C; 0.3%) for 10 days to induce malignant hypertension. Rats induced with I3C had higher mean arterial pressures (173 ± 9 vs. 112 ± 11 mmHg, P < 0.01) than noninduced normotensive rats ( n = 9). Glomerular damage was evaluated by determination of the glomerulosclerosis index (GSI) in tissue sections stained with periodic acid-Schiff. Kidneys of hypertensive rats had a higher GSI than normotensive rats (21.3 ± 5.6 vs. 3.5 ± 1.31 units). Quantitative analysis of macrophage ED-1-positive cells and proliferating cell nuclear antigen using immunohistochemistry demonstrated increased macrophage numbers in the renal interstitium (106.4 ± 11.4 vs. 58.7 ± 5.0 cells/mm2) and increased proliferating cell number in cortical tubules (37.8 ± 5.7 vs. 24.2 ± 2.1 cells/mm2), renal cortical vessels (2.2 ± 0.5 vs. 0.13 ± 0.07 cells/vessel), and the cortical interstitium (33.6 ± 5.7 vs. 4.2 ± 1.4 cells/mm2) of hypertensive rat kidneys. These findings demonstrate that the renal pathological changes that occur during the development of malignant hypertension in Cyp1a1-Ren2 rats are characterized by inflammation and cellular proliferation in cortical vessels and tubulointerstitium.

113 INTERACTIONS BETWEEN THE VISCERAL YOLK SAC PLACENTA AND THE UTERINE WALL IN NECROMYS LASIURUS (RODENTIA, CRICETIDAE)—AN EXPERIMENTAL MODEL TO UNDERSTANDING THE MATERNAL–FETAL EXCHANGE

2012 ◽  
Vol 24 (1) ◽  
pp. 169
Author(s):  
P. O. Favaron ◽  
A. M. Carter ◽  
A. M. Mess ◽  
M. F. Oliveira ◽  
M. A. Miglino
Keyword(s):  
Periodic Acid ◽  
Uterine Wall ◽  
Yolk Sac ◽  
Laboratory Animals ◽  
Nuclear Antigen ◽  
Fetal Membrane ◽  
Strong Response ◽  
Periodic Acid Schiff ◽  
Vital Functions ◽  
Visceral Yolk Sac

The establishment of pregnancy results from the interaction between the trophoblast and maternal tissues. Evolutionarily, the yolk sac (YS) is the only fetal membrane present in all vertebrate taxa. In mammals, it results in the development of the YS placenta, which is responsible for maternal–fetal exchange in the beginning of gestation. Its functions are related to hematopoiesis, selective transfer of various substances including proteins, iron transfer and histiotrophic nutrition. Necromys lasiurus is a rodent species from South America, which is related to species which are used as laboratory animals. Herein, we show data on the early development of the YS to promote a better understanding of its relationship with the uterine wall. Ten implantation sites of Necromys were obtained from a breeding group at the UFERSA, Mossoró, Brazil. Samples were examined by means of histology, immunohistochemistry for vimentin (1:200) and Proliferating Cell Nuclear Antigen (PCNA; 1:800) and scanning and transmission electron microscopy. In early pregnancy the visceral YS was in places closely attached to the uterine wall. At these attachments, the uterus possessed infolded structures with a high-columnar epithelium associated with blood vessels and surrounded by vimentin positive glands. In these areas, the YS became compact in shape and we observed an intimate association of both visceral yolk sac and endometrial luminal epithelium. A strong response to periodic acid Schiff, including plenty of granular vesicles, indicated secretory activity of the endodermal cells, such as release of histiotrophe. Large intercellular spaces, vacuoles and glycogen were present in the cytoplasm of these cells. On the other hand, in areas where the visceral YS was near to the labyrinthine region of chorioallantoic placenta, it was highly villous. The villi were formed by endoderm cells with hexagonal shape. The visceral YS was supplied by fetal vessels, which form the blood islands, structures related to the fetal hematopoiesis. A prominent microvilli area was observed on the surface of the endoderm cells. Additionally, vacuoles, electron-dense inclusions and dense droplets indicative of high secretory and transfer activities were seen inside of these cells. According to PCNA labelling, the mesothelial layer in the region near to uterine consisted of proliferative cells. Although early gestation represents a crucial developmental stage, information about the interaction between the trophoblast and maternal tissues is sparse. Embryonic mortality, a major cause of reproductive failure in livestock species, can be related to interactive problems between maternal and fetal tissues that affect exchange of nutrients, hematopoiesis and other vital functions. Supported by grants from FAPESP (Proc. 09/53392-8).

scholarly journals Production of capsular material by equine trophoblast transplanted into immunodeficient mice

Reproduction ◽  
2003 ◽  
pp. 855-863 ◽  
Author(s):  
A Albihn ◽  
RO Waelchli ◽  
J Samper ◽  
JG Oriol ◽  
BA Croy ◽  
...  
Keyword(s):  
Periodic Acid ◽  
Endometrial Biopsy ◽  
Specific Staining ◽  
Periodic Acid Schiff ◽  
In Vitro Techniques ◽  
Immunodeficient Mice ◽  
Xenogeneic Transplantation ◽  
Pas Staining ◽  
Capsular Material

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

scholarly journals The role of histopathology in the diagnosis of dermatophytoses / Značaj patohistološkog nalaza u dijagnostici dermatofitoza

2010 ◽  
Vol 2 (2) ◽  
pp. 45-53 ◽  
Author(s):  
Đorđi Gocev ◽  
Katerina Damevska
Keyword(s):  
Fungal Infection ◽  
Periodic Acid ◽  
False Negative ◽  
Histopathological Analysis ◽  
Unexpected Finding ◽  
Skin Infections ◽  
Specific Staining ◽  
Periodic Acid Schiff ◽  
Skin Reactions ◽  
Pas Staining

Abstract Histopathological analysis is not a routine procedure for diagnosing fungal skin infections. In the histopathological specimens, fungi are visible only when using special stain such as periodic acid-Schiff (PAS). However, histopathological analysis may not be performed in small laboratories. Histopathological characteristics of fungal skin infections are not specific. In all skin biopsy cases, obtained without clinical suspicion of fungal infection, the knowledge of certain, most frequent histopathological reaction patterns, as well as specific histopathological indicators (a diagnostic histopathological “clue”), of certain superficial mycoses e.g., dermatophytoses, may raise a suspicion of fungal infection and warrant a fungal-specific staining. A retrospective analysis of all PAS-stained sections was carried out. All PAS-positive biopsy specimens were assessed for clinical features, histopathological patterns of skin reactions, and presence of histopathological indicators. Our results have shown that out of the total of 361 PAS-stained sections, fungal hyphae were identified in 12 (3.3%) specimens. In 5 (1.4%) cases, the diagnosis of fungal infection was suspected on clinical grounds, while in 7 (1.9%) cases detection of fungi was an unexpected finding. The most frequent type of histopathological pattern was spongiotic, and the most frequent histopathological indicator was the presence of neutrophils within the epidermis. Our results confirm that dermatophytoses may present with clinical and histological non-specific findings. PAS staining represents a relatively cheap and simple fungal-specific staining. It has been suggested that it not only confirms that the selected material is actually invaded, but also reduces the number of false-negative direct reports, where fungi are cultured from a microscopically negative specimen. Apart from a small percentage of positive findings, our results justify the need for routine PAS staining of all clinically and histologically non-specific inflammatory skin conditions.

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