Partial formation of sperm dimorphism from spermatocytes of the cottoid fish, Hemilepidotus gilberti in cell culture

SummaryPolymorphism of sperm is considered to be significant for the reproductive strategy in some animal species. The phenomenon is thought to occur in the species-specific stage of spermatogenesis, but how the identical germ cells are differentiated towards polymorphic sperm remains unknown. We here performed a germ cell culture in the cottoid fish, Hemilepidotus gilberti, whose sperm exhibit dimorphism with fertilizable eusperm and unfertilizable parasperm. In the culture, germ cells, which were obtained with an identical morphology, a spherical shape of 5–7 µm in diameter, differentiated into smaller spherical cells with a single nucleus, a moving flagellum and localized mitochondria. In addition, large retroflex-shaped cells with two elongated nuclei were also observed in the cell culture. Germ cells that had each morphological feature were histologically also observed in some cysts of the spermatogenetic testis, suggesting that the former type of cell corresponded to developing eusperm and the latter corresponded to developing parasperm. When BrdU was incorporated into germ cells in the culture, it was detected in both cells with eusperm-like and those with parasperm-like morphologies. These findings suggest that DNA-duplicating spermatocytes are potent to autonomously progress a part of spermatogenesis to form dimorphic sperm.

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Comparative study of adenoviruses with monoclonal antibodies

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651992000100004 ◽

1992 ◽

Vol 34 (1) ◽

pp. 19-26 ◽

Cited By ~ 2

Author(s):

Terezinha Maria de Paiva ◽

Sueko Takimoto ◽

María Akíko Ishida ◽

María Candida Oliveira de Souza ◽

Tuneo Ishimaru ◽

...

Keyword(s):

Cell Culture ◽

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Comparative Study ◽

Neutralization Test ◽

Human Adenovirus ◽

Enzyme Linked Immunosorbent Assay ◽

Viral Neutralization ◽

Species Specific ◽

Viral Neutralization Test

The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII) were obtained.

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Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1483-1486.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1483-1486 ◽

Cited By ~ 33

Author(s):

Hui Wang ◽

Fanrong Kong ◽

Peter Jelfs ◽

Gregory James ◽

Gwendolyn L. Gilbert

Keyword(s):

Cell Culture ◽

16S Rrna ◽

Cell Line ◽

Intergenic Spacer ◽

23S Rrna ◽

Pcr Assay ◽

Intergenic Spacer Region ◽

Specific Pcr ◽

Specific Pcr Assay ◽

Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

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A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad

Pathogens ◽

10.3390/pathogens10111431 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1431

Author(s):

Karla Georges ◽

Chuckwudozi Ezeokoli ◽

Godwin Isitor ◽

Alex Mutani ◽

Olivier Sparagano ◽

...

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Inclusion Bodies ◽

Clinical Signs ◽

Reverse Line Blot ◽

Rrna Gene ◽

Canine Ehrlichiosis ◽

Autologous Cell ◽

Blot Hybridisation ◽

Species Specific

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

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Conserved cell types with divergent features between human and mouse cortex

10.1101/384826 ◽

2018 ◽

Cited By ~ 16

Author(s):

Rebecca D Hodge ◽

Trygve E Bakken ◽

Jeremy A Miller ◽

Kimberly A Smith ◽

Eliza R Barkan ◽

...

Keyword(s):

Rna Sequencing ◽

Cognitive Abilities ◽

Cell Types ◽

Mouse Cell ◽

Middle Temporal Gyrus ◽

Cellular Architecture ◽

Mouse Cortex ◽

Single Nucleus ◽

Species Specific ◽

Human And Mouse

AbstractElucidating the cellular architecture of the human neocortex is central to understanding our cognitive abilities and susceptibility to disease. Here we applied single nucleus RNA-sequencing to perform a comprehensive analysis of cell types in the middle temporal gyrus of human cerebral cortex. We identify a highly diverse set of excitatory and inhibitory neuronal types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to a similar mouse cortex single cell RNA-sequencing dataset revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of human cell type properties. Despite this general conservation, we also find extensive differences between homologous human and mouse cell types, including dramatic alterations in proportions, laminar distributions, gene expression, and morphology. These species-specific features emphasize the importance of directly studying human brain.

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028. Cytokine networks and regulation of spermatogenesis - what should we really believe?

Reproduction Fertility and Development ◽

10.1071/srb05abs028 ◽

2005 ◽

Vol 17 (9) ◽

pp. 70

Author(s):

M. P. Hedger

Keyword(s):

Growth Factors ◽

Germ Cells ◽

Sertoli Cells ◽

Seminiferous Epithelium ◽

Specific Rate ◽

Development In Vitro ◽

Cytokine Networks ◽

Species Specific ◽

Key Events

Spermatogenesis is a complex yet highly organised process involving intimate interactions between the supporting Sertoli cells and germ cells at various stages of development. The repeating pattern of the cycle of the seminiferous epithelium is due to the fact that spermatogonia enter spermatogenesis at regularly spaced intervals and proceed through the process at a species-specific rate. How this degree of coordination is maintained remains poorly understood, but recent evidence has focussed attention on the role of growth factors produced by the Sertoli cells and germ cells. Several of these growth factors, such as interleukin-1α (IL-1α), IL-6, tumour necrosis factor (TNFα) and activin A, are also inflammatory cytokines. This has led some researchers to question the physiological significance of these data with respect to normal testicular function. For example, in spite of the fact that IL-1α is produced by the Sertoli cell and regulates spermatogonial proliferation and development in vitro, mice lacking the IL-1R, and hence unresponsive to IL-1α, possess relatively normal fertility. So what role, if any, do these cytokines play in the normal testis, or are they only important during inflammation? It is quite evident that these cytokines have stimulatory and/or inhibitory effects on spermatogonial and spermatocyte development. These cytokines also interact at multiple levels within each other’s signalling pathways and have considerable redundancy of action. Moreover, expression of these cytokines varies across the cycle of the seminiferous epithelium, with major changes in production coinciding with two key events within the cycle: the release of sperm from the epithelium, and the major peaks of DNA synthesis by spermatogonia and preleptotene spermatocytes. It is therefore possible to hypothesise that release of sperm and resorption of the residual cytoplasm triggers a self-regulating inflammatory cascade within the epithelium that initiates and then modulates the next round of spermatogenic development, ensuring that spermatogonia enter the process at the appropriately spaced intervals.

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Response to fibronectin of mouse primordial germ cells before, during and after migration

Development ◽

10.1242/dev.113.4.1365 ◽

1991 ◽

Vol 113 (4) ◽

pp. 1365-1373 ◽

Cited By ~ 6

Author(s):

C. Ffrench-Constant ◽

A. Hollingsworth ◽

J. Heasman ◽

C.C. Wylie

Keyword(s):

Cell Culture ◽

Extracellular Matrix ◽

Germ Cells ◽

Culture System ◽

Primordial Germ Cells ◽

Explant Culture ◽

Double Labelling ◽

Target Tissue ◽

Fibronectin Mrna

The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.

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Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture

Development ◽

10.1242/dev.129.14.3359 ◽

2002 ◽

Vol 129 (14) ◽

pp. 3359-3365 ◽

Cited By ~ 1

Author(s):

Noriyoshi Sakai

Keyword(s):

Cell Culture ◽

In Vitro Fertilization ◽

Germ Cell ◽

Germ Cells ◽

Regulatory Function ◽

Feeder Cells ◽

Male Germ Cells ◽

Vitro Fertilization ◽

Testicular Cells

Because cell culture systems are easily accessible for experimental genetic manipulation, male germ cell culture is of great usefulness in creating sperm vectors. This report describes that cultured male germ cells of zebrafish (Danio rerio) underwent mitosis and transmeiotic differentiation, including the entire process of meiosis, to develop into functional sperm. Enzymatically dissociated testicular cells containing germ cells were co-cultured on feeder cells derived from tumor-like testis, which exhibited features characteristic of Sertoli cells such as phagocytic activity and transcription of the Wilms’ tumor suppressor wt1 and sox9a genes. Germ cells formed a clump, divided by mitosis, and differentiated into flagellated sperm on the feeders. Expression of the germ cell marker gene vas was prolonged in co-culture with the feeders, compared with culture of dissociated testicular cells alone, indicating that the feeder cells stimulate proliferation of spermatogonia. When cultured germ cells/sperm with the feeders were used for in vitro fertilization, normal embryos were obtained. Addition of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into culture medium resulted in BrdU-positive sperm and four-cell stage embryos after in vitro fertilization. This culture system should prove useful not only in producing transfected functional sperm, but also in analyzing the regulatory function of testicular somatic cells on the mitosis and meiosis of male germ cells in vertebrates.

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The Rhodnius testis: hormones, differentiation of the germ cells, and duration of the molting cycle

Canadian Journal of Zoology ◽

10.1139/z75-202 ◽

1975 ◽

Vol 53 (11) ◽

pp. 1673-1681 ◽

Cited By ~ 13

Author(s):

J. B. Dumser ◽

K. G. Davey

Keyword(s):

Cell Division ◽

Germ Cell ◽

Juvenile Hormone ◽

Germ Cells ◽

Fourth Instar ◽

Fourth Stage ◽

Molting Cycle ◽

Male Germ Cells ◽

Germ Cell Development ◽

Species Specific

Allatectomy of third- and fourth-stage Rhodnius larvae results in the production of precocious adults at the succeeding molt. When the allatectomy is performed on fourth-instar larvae, the testes of which contain spermatocysts of approximately 26 cells, spermatozoa are evident in the testis of the resulting precocious adult. If the allatectomy is performed on third-instar larvae, the testes of which contain spermatocysts of approximately 24 cells, the precocious adult never shows germ cell development beyond the early spermatocyte (28) stage, in spite of extensive metamorphosis of mesodermal and ectodermal structures. These results support the view that differentiation of the male germ cells in insects is inflexibly tied to a species-specific division sequence, and thus not directly manipulable by morphogenetic hormones. Evidence is also presented that the presence or absence of juvenile hormone influences the duration of the molt and hence the available time for germ cell division within each instar.

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In-Vitro Cell Culture for Efficient Assessment of Mycotoxin Exposure, Toxicity and Risk Mitigation

Toxins ◽

10.3390/toxins12030146 ◽

2020 ◽

Vol 12 (3) ◽

pp. 146 ◽

Cited By ~ 3

Author(s):

Ran Xu ◽

Niel A. Karrow ◽

Umesh K. Shandilya ◽

Lv-hui Sun ◽

Haruki Kitazawa

Keyword(s):

Cell Culture ◽

Animal Feed ◽

Risk Mitigation ◽

Mitigation Strategies ◽

Initial Assessment ◽

In Vitro Cell Culture ◽

Culture Models ◽

Species Specific ◽

By Products

Mycotoxins are toxic secondary fungal metabolites that commonly contaminate crops and food by-products and thus, animal feed. Ingestion of mycotoxins can lead to mycotoxicosis in both animals and humans, and at subclinical concentrations may affect animal production and adulterate feed and animal by-products. Mycotoxicity mechanisms of action (MOA) are largely unknown, and co-contamination, which is often the case, raises the likelihood of mycotoxin interactions. Mitigation strategies for reducing the risk of mycotoxicity are diverse and may not necessarily provide protection against all mycotoxins. These factors, as well as the species-specific risk of toxicity, collectively make an assessment of exposure, toxicity, and risk mitigation very challenging and costly; thus, in-vitro cell culture models provide a useful tool for their initial assessment. Since ingestion is the most common route of mycotoxin exposure, the intestinal epithelial barrier comprised of epithelial cells (IECs) and immune cells such as macrophages, represents ground zero where mycotoxins are absorbed, biotransformed, and elicit toxicity. This article aims to review different in-vitro IEC or co-culture models that can be used for assessing mycotoxin exposure, toxicity, and risk mitigation, and their suitability and limitations for the safety assessment of animal foods and food by-products.

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Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes

Scientific Reports ◽

10.1038/s41598-020-64977-3 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

D. Dedukh ◽

S. Riumin ◽

M. Chmielewska ◽

B. Rozenblut-Kościsty ◽

K. Kolenda ◽

...

Keyword(s):

Germ Cells ◽

Parental Species ◽

Genetic Material ◽

Single Chromosome ◽

Water Frog ◽

Genome Reorganization ◽

Interspecies Hybrids ◽

Metaphase Stage ◽

Pelophylax Esculentus ◽

Species Specific

Abstract In most organisms, cells typically maintain genome integrity, as radical genome reorganization leads to dramatic consequences. However, certain organisms, ranging from unicellular ciliates to vertebrates, are able to selectively eliminate specific parts of their genome during certain stages of development. Moreover, partial or complete elimination of one of the parental genomes occurs in interspecies hybrids reproducing asexually. Although several examples of this phenomenon are known, the molecular and cellular processes involved in selective elimination of genetic material remain largely undescribed for the majority of such organisms. Here, we elucidate the process of selective genome elimination in water frog hybrids from the Pelophylax esculentus complex reproducing through hybridogenesis. Specifically, in the gonads of diploid and triploid hybrids, but not those of the parental species, we revealed micronuclei in the cytoplasm of germ cells. In each micronucleus, only one centromere was detected with antibodies against kinetochore proteins, suggesting that each micronucleus comprises a single chromosome. Using 3D-FISH with species-specific centromeric probe, we determined the role of micronuclei in selective genome elimination. We found that in triploid LLR hybrids, micronuclei preferentially contain P. ridibundus chromosomes, while in diploid hybrids, micronuclei preferentially contain P. lessonae chromosomes. The number of centromere signals in the nuclei suggested that germ cells were aneuploid until they eliminate the whole chromosomal set of one of the parental species. Furthermore, in diploid hybrids, misaligned P. lessonae chromosomes were observed during the metaphase stage of germ cells division, suggesting their possible elimination due to the inability to attach to the spindle and segregate properly. Additionally, we described gonocytes with an increased number of P. ridibundus centromeres, indicating duplication of the genetic material. We conclude that selective genome elimination from germ cells of diploid and triploid hybrids occurs via the gradual elimination of individual chromosomes of one of the parental genomes, which are enclosed within micronuclei.

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