The human cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG), as well as the widely consumed plant (phyto)cannabinoid Δ9-tetrahydrocannabinol (THC)1. The cannabinoid receptors have been the targets of intensive drug discovery efforts due to the therapeutic potential of modulators for controlling pain2, epilepsy3, obesity4, and other maladies. While much progress has recently been made in understanding the biophysical properties of GPCRs, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. We used GPCR engineering and lipidic cubic phase (LCP) crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6 Å resolution. CB1's extracellular surface, including the highly conserved membrane-proximal amino-terminal (N-terminal) region, is distinct from other lipid-activated GPCRs and forms a critical part of the ligand binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function, and will aid the design and optimization of cannabinoid system modulators for therapeutic ends.

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.germ line ͉ spermatogenesis ͉ gene targeting ͉ enhanced GFP fluorescence T he laboratory rat represents one of the most comprehensively studied mammalian species, with described use in Ͼ1 million publications in a wide range of medically relevant areas. Qualities such as size, fecundity, behavior, ease of surgical techniques, tissue sampling, and general laboratory management have contributed to its popularity (1-3). However, a failure to develop technology to produce rat genetic models through gene targeting has resulted in the mouse becoming a widely popular animal model.Although mouse embryonic stem (ES) cells renew with a sense of immortality, primitive hemapoietic stem cells self renew ineffectively and for only a short period in vitro (4, 5). ES cells from species other than the mouse or human fail to generally self renew effectively and also lose pluripotency in culture (6). We and others have not succeeded in culturing pluripotent ES cells from the rat; however, if spermatogonial stem cells could be cultured under conditions where they self renewed and expanded in numbers, conceivably they could also be genetically modified in vitro in much the same manner as seen with mouse ES cells. This alternative to the use of genetically modified ES cells would result in direct germ line transfer and an escape from the intervening formation of a mosaic animal. After appropriate selection of gene-targeted cells in culture, the chosen spermatogonial stem cells could either be induced to differentiate to the haploid stage in vitro or transplanted to the testes of recipient rats to allow development to the haploid stage. In either case, intracytoplasmic sperm injection into the egg would result in transmission of the genetically modified information (7,8).We now show that genetically marked spermatogonial stem cells from transgenic germ-cell-specific EGFP (GCS-EGFP) rats (9) can be placed under defined culture co...

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 ≈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 ≈50% of pups in the F 2 generation. Thus, by using these procedures, ≈30% of pups in the F 1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

No abstract

The spermatogenesis and oogenesis-specific transcription factor Sohlh2 is normally expressed only in premeiotic germ cells. In this study, Sohlh2 and several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that overexpresses bone morphogenetic protein 4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile because of a block in spermatogenesis. Although normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type B spermatogonia by postnatal day 7. By day 10, development to the preleptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type A spermatogonia (DAZL؉/ PLZF؉) to differentiating type A spermatogonia (DAZL؉/ PLZF؊) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice proliferated normally but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase cKit. Thus, Sohlh2 is required for progression of differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis. STEM CELLS 2008;26: 1587-1597 Disclosure of potential conflicts of interest is found at the end of this article.

Disrupting genes in the rat on a genome-wide scale will allow the investigation of many biological processes linked to human health. Here we used transposon-mediated mutagenesis to knock out genes in rat spermatogonial stem cells. Given the capacity of the testis to support spermatogenesis from thousands of transplanted, genetically manipulated spermatogonia, this approach paves a way for high-throughput functional genomic studies in the laboratory rat.

An economical and simplified procedure to derive and propagate fully functional lines of undifferentiated rat spermatogonia in vitro is presented. The procedure is based on the formulation of a new spermatogonial culture medium termed SG medium. The SG medium is composed of a 1:1 mixture of Dulbecco modified Eagle medium:Ham F12 nutrient, 20 ng/ml of GDNF, 25 ng/ml of FGF2, 100 microM 2-mercaptoethanol, 6 mM l-glutamine, and a 1x concentration of B27 Supplement Minus Vitamin A solution. Using SG medium, six individual spermatogonial lines were derived from the testes of six separate Sprague-Dawley rats. After proliferating over a 120-day period in SG medium, stem cells within the spermatogonial cultures effectively regenerated spermatogenesis in testes of busulfan-treated recipient rats, which transmitted the donor cell haplotype to more than 75% of progeny by natural breeding. Subculturing in SG medium did not require protease treatment and was achieved by passaging the loosely bound spermatogonial cultures at 1:3 dilutions onto fresh monolayers of irradiated DR4 mouse fibroblasts every 12 days. Spermatogonial lines derived and propagated using SG medium were characterized as homogeneous populations of ZBTB16(+) DAZL(+) cells endowed with spermatogonial stem cell potential.

SUMMARY Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate “pure”, non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetically selecting donor spermatogonia. Monoclonal enrichment of Erbb3-null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform to generate targeted germline mutations in rats, and to study spermatogenesis.