Biomedical subjects

F Kent Hamra

Publications and source records attributed to F Kent Hamra.

5 recordsLinked to original sources

PubMed2005

Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture.

The use of a transgenic line of rats that express enhanced GFP (EGFP) exclusively in the germ line has allowed a separation of feeder layers and contaminating testis somatic cells from germ cells and the identification of a set of spermatogonial stem cell marker transcripts. With these molecular markers as a guide, we have now devised culture conditions where rat spermatogonial stem cells renew and proliferate in culture with a doubling time between 3 and 4 days. The marker transcripts increase in relative abundance as a function of time in culture, and the stem cells retain competency to colonize and develop into spermatids after transplantation to the testes of recipient rats. The cells also remain euploid after at least 12 passages. Cell lines could be isolated and cryopreserved and, upon subsequent thawing, continue to self renew. Transfection of the spermatogonial stem cells with a plasmid containing the neomycin phosphotransferase (neo) selectable marker resulted in selection of G418-resistant cell lines that effectively colonize recipient testes, suggesting that gene targeting is now feasible in the rat.

Animals↗

PubMed2005

Expression patterns of mitotic and meiotic cell cycle regulators in testicular cancer and development.

Mitotic and meiotic cell cycle regulation is essential for normal development and tumor prevention. The underlying molecular mechanisms are not completely characterized. The aim of our analysis was to derive a global expression map of cell cycle regulators in mitosis and meiosis. First, the expression of cyclins, CDKs and CDK inhibitors was determined during postnatal testis maturation in mice using microarrays and quantitative RT-PCR. The abundance of cyclins A1, B2, K, M4, CDK2, all CDKLs, CDKN2c, CDKN2d and INCA1 increased during testis maturation. In contrast, cyclins A2, B1, D2, G1, G2, CDK1, CDK4 and CDK2AP1 showed a maturation-associated decrease. Gene expression profiles of isolated germ cells and testicular somatic cells confirmed these results. Second, we determined cyclin expression patterns in human normal and malignant testis samples (n = 36) modeling the reciprocal difference between meiosis and mitosis. Testicular tumors strictly expressed cell cycle regulators identified in mitotically dividing germ cells. Expression of several transcripts was histology-specific in testicular tumors, providing novel molecular markers and potential therapeutic targets. Taken together, our data provide a comprehensive expression map of cell cycle regulators at the switch between mitosis and meiosis in testis development and in cancerogenesis.

Animals↗

PubMed2004

Defining the spermatogonial stem cell.

Through the use of donor cells from transgenic rats expressing GFP exclusively in the germline, we have defined culture conditions where male germ cells lose (on STO cells) or maintain (on MSC-1 cells) stem cell activity. A cadre of germ cell transcripts strikingly decrease in relative abundance as a function of testis age or culture time on STO cells, but only a subset of these transcripts (approximately 248) remain elevated when cultured on MSC-1 cells. If specific gene expression regulates stem cell activity, some or all of these transcripts are candidates as such regulators. We establish a spermatogonial stem cell index (SSCI) that reliably predicts relative stem cell activity in rat or mouse testis cell cultures, and through the use of an antibody to a robust signal (Egr3) within the index find intense signals in single or paired cells. As germ cells form longer interconnected chains (incomplete cytokinesis), the Egr3 signal disappears coincident with a loss of stem cell activity. Thus, molecular markers specific for spermatogonial stem cells establish a reliable and rapid means by which to define these cells in culture and alleviate the need for laborious testicular transfers in initial cell culture studies.

Animals↗

PubMed2003

A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.

Understanding mammalian spermatozoan development and the events surrounding fertilization has grown slowly, in part because of uncertainty about the number and identity of the cellular components involved. Determination of those transcripts expressed specifically by germ cells should provide an inclusive list of probable critical proteins. Here, total mouse testis transcript profiles were trimmed of transcripts found in cultures enriched in Sertoli or interstitial cells to yield a germ cell-enriched transcript profile. Monitoring of changes of this profile in the developing testis identified 1,652 genes whose transcript abundance increased markedly coincident with the onset of meiosis. Remarkably, 351 of these genes (approximately equal to 20%) appear to be expressed only in the male germline. Germ cell-specific transcripts are much less common earlier in testis development. Further analysis of the UniGene EST database coupled with quantitative PCR indicates that approximately 4% of the mouse genome is dedicated to expression in postmeiotic male germ cells. Most or many of the protein products of these transcripts are probably retained in mature spermatozoa. Targeted disruption of 19 of these genes has indicated that a majority have roles critical for normal fertility. Thus, we find an astonishing number of genes expressed specifically by male germ cells late in development. This extensive group provides a plethora of potential targets for germ cell-directed contraception and a staggering number of candidate proteins that could be critical for fertilization.

Animals↗

PubMed2002

Production of transgenic rats by lentiviral transduction of male germ-line stem cells.

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin. The cells that bound to laminin represented approximately 5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to approximately 50% of pups in the F(2) generation. Thus, by using these procedures, approximately 30% of pups in the F(1) generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

Amino Acid Sequence↗