Radioprotection of beta-carotene evaluated on mouse somatic and germ cells

In the present paper, the protective effect of beta-carotene was evaluated after whole body exposure of mice to 2 Gy of X-rays. Splenocytes, reticulocytes, bone marrow cells and spermatids were evaluated for the frequency of micronuclei (MN) induced by X-rays. Mice were treated (gavage) with beta-carotene (10, 25 and 50 mg/kg b.w.) for 5 consecutive days and, 4 h after the last treatment, the animals were irradiated. The results obtained showed different frequencies of X-ray-induced-MN between different cell populations analysed and also different response of these cells to the beta-carotene treatment. The radioprotective effect of beta-carotene was observed in splenocytes, reticulocytes, and spermatids but not in bone marrow cells. No dose-response relationship for beta-carotene was detected. The time of sampling, the sensitivity of the cells as well as the antioxidant activity of beta-carotene are discussed as important factors for the radioprotective action of this provitamin.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytoplasmic bridges, intercellular junctions, and individualization of germ cells during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

During mitotic and meiotic divisions in Dermatobia hominis spermatogenesis, the germ cells stay interlinked by cytoplasm, bridges as a result of incomplete cytokinesis. By the end of each division, cytoplasmic bridges flow to the center of the cyst, forming a complex, called the fusoma. During meiotic prophase I, spermatocytes I present desmosome-like junctions and meiotic cytoplasmic bridges. At the beginning of spermiogenesis, the fusoma moves to the future caudal end of the cyst, and at this time the early spermatids are linked by desmosome-like junctions. Throughout spermiogensis, new and sometimes broad cytoplasmic bridges are formed among spermatids at times making them share cytoplasm. In this case the individualization of cells is assured by the presence of smooth cisternae that outline then structures The more differentiated spermatids have in addition to narrow cytoplasmic bridges, plasmic membranes junctions. By the end of spermiogenesis the excess cytoplasmic mass is eliminated leading to spermatid individualization. Desmosome-like junctions of spermatocytes I and early spermatids appear during the fusoma readjustment and segregations; on the other hand, plasmic membrane junctions appear in differentiating spermatids and are eliminated along with the cytoplasmic excess. These circumstances suggest that belt desmosome-like and plasmic membrane junctions are involved in the maintenance of the relative positions of male germ cells in D. hominis while they are inside the cysts. © 1996 Wiley-Liss, Inc.

Automatic classification of fish germ cells through optimum-path forest

The spermatogenesis is crucial to the species reproduction, and its monitoring may shed light over some important information of such process. Thus, the germ cells quantification can provide useful tools to improve the reproduction cycle. In this paper, we present the first work that address this problem in fishes with machine learning techniques. We show here how to obtain high recognition accuracies in order to identify fish germ cells with several state-of-the-art supervised pattern recognition techniques. © 2011 IEEE.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Disperse Red 1 (textile dye) induces cytotoxic and genotoxic effects in mouse germ cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Centriole behaviour during meiosis of male germ cells of Dermatobia hominis (Diptera:Cuterebridae)

During the meiotic division of Dermatobia hominis spermatogenesis, the centrioles duplicate only in prophase I, giving rise to short cilia which are exposed on the cellular surface. In metaphase I they are internalized and distributed to the daughter cells. Consequently, the secondary spermatocytes have two centrioles which repeat the cycle of cilia externalization followed by internalization. The spermatids receive only one centriole, which changes into a basal body and originates a flagellum. This centriole behaviour seems to be a general feature in insect male germ cell meiosis.

Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.

Derivation of pluripotent stem cells from cultured human primordial germ cells

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Highly efficient base excision repair (BER) in human and rat male germ cells

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.