Spermatogonial Stem Cells (SSCs) in Buffalo (Bubalus bubalis) Testis

BACKGROUND: Water buffalo is an economically important livestock species and about half of its total world population exists in India. Development of stem cell technology in buffalo can find application in targeted genetic modification of this species. Testis has emerged as a source of pluripotent stem cells in mice and human; however, not much information is available in buffalo. OBJECTIVES AND METHODS: Pou5f1 (Oct 3/4) is a transcription factor expressed by pluripotent stem cells. Therefore, in the present study, expression of POU5F1 transcript and protein was examined in testes of both young and adult buffaloes by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Further, using the testis transplantation assay, a functional assay for spermatogonial stem cells (SSCs), stem cell potential of gonocytes/spermatogonia isolated from prepubertal buffalo testis was also determined. RESULTS: Expression of POU5F1 transcript and protein was detected in prepubertal and adult buffalo testes. Western blot analysis revealed that the POU5F1 protein in the buffalo testis exists in two isoforms; large (∼47 kDa) and small (∼21 kDa). Immunohistochemical analysis revealed that POU5F1 expression in prepubertal buffalo testis was present in gonocytes/spermatogonia and absent from somatic cells. In the adult testis, POU5F1 expression was present primarily in post-meiotic germ cells such as round spermatids, weakly in spermatogonia and spermatocytes, and absent from elongated spermatids. POU5F1 protein expression was seen both in cytoplasm and nuclei of the stained germ cells. Stem cell potential of prepubertal buffalo gonocytes/spermatogonia was confirmed by the presence of colonized DBA-stained cells in the basal membrane of seminiferous tubules of xenotransplanted mice testis. CONCLUSION/SIGNIFICANCE: These findings strongly indicate that gonocytes/spermatogonia, isolated for prepubertal buffalo testis can be a potential target for establishing a germ stem cell line that would enable genetic modification of buffaloes

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

Background: Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. Methodology/Principal Findings: Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. Conclusion: Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserv

The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome

Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish

Background Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called “niche” which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate. Methodology/Principal Findings Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5′-bromo-2′-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish. Conclusions/Significance We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 bp), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of CDSs encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. T. crunogena XCL-2 is unusual among obligate sulfur oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. A 38 kb prophage is present, and a high level of prophage induction was observed, which may play a role in keeping competing populations of close relatives in check. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome

Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models

La historia de la fisioterapia se debe entender desde el conjunto de actuaciones, métodos y técnicas que de forma personal o colectiva se han basado en el empleo de los agentes físicos de situaciones histórico-social diferentes con el fin de promover la salud. De tal forma y en consecuencia, tanto con el avance de las herramientas con que cuenta el fisioterapeuta, con la diversidad de patologías y lesiones, resulta relevante e importante conocer la manera correcta e indicada de utilizar diferentes medios físicos, mecánicos, eléctricos y manuales; así como también contraindicaciones, cuidados y medidas de seguridad a fin de evitar y/o prevenir daños o lesiones en los pacientes.Tener acceso a la plataforma Moodl