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

Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes

Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells

The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment

Substantial improvements have been made in recent years in the ability to engraft human cells and tissues into immunodeficient mice. The use of human hematopoietic stem cells (HSCs) leads to multi-lineage human hematopoiesis accompanied by production of a variety of human immune cell types. Population of murine primary and secondary lymphoid organs with human cells occurs, and long-term engraftment has been achieved. Engrafted cells are capable of producing human innate and adaptive immune responses, making these models the most physiologically relevant humanized animal models to date. New models have been successfully infected by a variety of strains of Human Immunodeficiency Virus Type 1 (HIV-1), accompanied by virus replication in lymphoid and non-lymphoid organs, including the gut-associated lymphoid tissue, the male and female reproductive tracts, and the brain. Multiple forms of virus-induced pathogenesis are present, and human T cell and antibody responses to HIV-1 are detected. These humanized mice are susceptible to a high rate of rectal and vaginal transmission of HIV-1 across an intact epithelium, indicating the potential to study vaccines and microbicides. Antiviral drugs, siRNAs, and hematopoietic stem cell gene therapy strategies have all been shown to be effective at reducing viral load and preventing or reversing helper T cell loss in humanized mice, indicating that they will serve as an important preclinical model to study new therapeutic modalities. HIV-1 has also been shown to evolve in response to selective pressures in humanized mice, thus showing that the model will be useful to study and/or predict viral evolution in response to drug or immune pressures. The purpose of this review is to summarize the findings reported to date on all new humanized mouse models (those transplanted with human HSCs) in regards to HIV-1 sexual transmission, pathogenesis, anti-HIV-1 immune responses, viral evolution, pre- and post-exposure prophylaxis, and gene therapeutic strategies