Packaging the male germline in plants

The development of plant lateral organs is interesting because, although many of the same genes seem to be involved in the early growth of primordia, completely different gene combinations are required for the complete development of organs such as leaves and stamens. Thus, the genes common to the development of most organs, which generally form and polarize the primordial 'envelope', must at some stage interact with those that 'install' the functional content of the organ--in the case of the stamen, the four microsporangia. Although distinct genetic pathways of organ initiation, polarity establishment and setting up the reproductive cell line can readily be recognized, they do not occur sequentially. Rather, they are activated early and run in parallel. There is evidence for continuing crosstalk between these pathways.     

Chloroplast-to-Nucleus Signaling Regulates MicroRNA Biogenesis in Arabidopsis

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Postmeiotic sex chromatin in the male germline of mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.     

Genome reprogramming and small interfering RNA in the Arabidopsis germline

The movement of mobile small RNA signals between cells has garnered much interest over the last few years, and has recently been extended to germ cells during gamete development. Focusing on plants, we review mobile RNA signals that arise following reprogramming in the germline, and their effect on transposable element silencing on the one hand and on meiotic and apomictic germ cell fate on the other. A potential role for reprogramming and small RNA in hybrid formation and speciation is proposed.     

Long-term proliferation in culture and germline transmission of mouse male germline stem cells

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.     

Pluripotency of male germline stem cells

The ethical issues and public concerns regarding the use of embryonic stem (ES) cells in human therapy have motivated considerable research into the generation of pluripotent stem cell lines from non-embryonic sources. Numerous reports have shown that pluripotent cells can be generated and derived from germline stem cells (GSCs) in mouse and human testes during in vitro cultivation. The gene expression patterns of these cells are similar to those of ES cells and show the typical self-renewal and differentiation patterns of pluripotent cells in vivo and in vitro. However, the mechanisms underlying the spontaneous dedifferentiation of GSCs remain to be elucidated. Studies to identify master regulators in this reprogramming process are of critical importance for understanding the gene regulatory networks that sustain the cellular status of these cells. The results of such studies would provide a theoretical background for the practical use of these cells in regenerative medicine. Such studies would also help elucidate the molecular mechanisms underlying certain diseases, such as testicular germ cell tumors.     

Phf7 controls male sex determination in the Drosophila germline

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. Here we identify PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). We propose that PHF7 acts as a conserved epigenetic "reader" that activates the male germline sexual program.     

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or "niches", which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. Here, we investigate the formation of the testis GSC niche, or "hub", during the late stages of embryogenesis. By morphological and molecular criteria, we identify and follow the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad.     

Transposition and gene disruption in the male germline of the mouse

We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation‐site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)‐trap transposon vectors, were outcrossed to wild‐type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics. genesis 30:82–88, 2001. © 2001 Wiley‐Liss, Inc.     

Retrovirus-mediated modification of male germline stem cells in rats

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.     

Elevated mutation rates in the germline of Polkappa mutant male mice

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of DNA polymerase kappa (Polkappa(-/-)) deficient mice. The spontaneous mutation rate in homozygous Polkappa(-/-) males was significantly higher than in isogenic wild-type mice (Polkappa(+/+)), but the ESTR mutation spectrum in Polkappa(-/-) animals did not differ from that in Polkappa(+/+) males. We suggest that compromised translesion synthesis in Polkappa(-/-) mice may result in replication fork pausing which, in turn, may affect ESTR mutation rate.     

Chromatin reprogramming: gender equality during Arabidopsis germline differentiation

Large-scale histone H3 reprogramming during male germline differentiation is conserved between animals and plants. A new report now shows that histone H3 reprogramming also occurs in the female germline of the flowering plant Arabidopsis thaliana.