Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway.

Piwi-interacting RNAs (piRNAs) are essential for silencing of transposable elements in the germline, but their biogenesis is poorly understood. Here we demonstrate that MOV10L1, a germ cell-specific putative RNA helicase, is associated with Piwi proteins. Genetic disruption of the MOV10L1 RNA helicase domain in mice renders both MILI and MIWI2 devoid of piRNAs. Absence of a functional piRNA pathway in Mov10l1 mutant testes causes loss of DNA methylation and subsequent derepression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile owing to complete meiotic arrest. This mouse mutant expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins.

Inactivation of Nxf2 causes defects in male meiosis and age-dependent depletion of spermatogonia.

In eukaryotes, mRNA is actively transported from nucleus to cytoplasm by a family of nuclear RNA export factors (NXF). While yeast harbors only one such factor (Mex67p), higher eukaryotes encode multiple NXFs. In mouse, four Nxf genes have been identified: Nxf1, Nxf2, Nxf3, and Nxf7. To date, the function of mouse Nxf genes has not been studied by targeted gene deletion in vivo. Here we report the generation of Nxf2 null mutant mice by homologous recombination in embryonic stem cells. Nxf2-deficient male mice exhibit fertility defects that differ between mouse strains. One third of Nxf2-deficient males on a mixed (C57BL/6x129) genetic background exhibit meiotic arrest and thus are sterile, whereas the remaining males are fertile. Disruption of Nxf2 in inbred (C57BL/6J) males impairs spermatogenesis, resulting in male subfertility, but causes no meiotic arrest. Testis weight and sperm output in C57BL/6J Nxf2(-/Y) mice are sharply reduced. Mutant epididymal sperm exhibit diminished motility. Importantly, proliferation of spermatogonia in Nxf2(-/Y) mice is significantly decreased. As a result, inactivation of Nxf2 causes depletion of germ cells in a substantial fraction of seminiferous tubules in aged mice. These studies demonstrate that Nxf2 plays a dual function in spermatogenesis: regulation of meiosis and maintenance of spermatogonial stem cells.

Abnormal sperm in mice lacking the Taf7l gene.

TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here, we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells by using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l(-/Y) mice, the weight of Taf7l(-/Y) testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology, including folded tails. Sperm motility was significantly reduced, and Taf7l(-/Y) males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l(-/Y) testes decreased more than twofold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ cell differentiation. Our mouse studies suggest that mutations in the human TAF7L gene might be implicated in X-linked oligozoospermia in men.

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse.

BACKGROUND: Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells - undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into As (single), Apr (paired), and Aal (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions. RESULTS: In the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (As, Apr, and Aal) in mouse. Ngn3 also specifically labels undifferentiated spermatogonia. We used Ngn3-GFP knockin mice, in which GFP expression is under the control of all Ngn3 transcription regulatory elements. Remarkably, Ngn3-GFP is only expressed in approximately 40% of LIN28-positive As (single) cells. The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia. CONCLUSION: Our study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations: Ngn3-GFP-negative (high stem cell potential) and Ngn3-GFP-positive (high differentiation commitment). Furthermore, Ngn3-GFP-negative cells are found in chains of Ngn3-GFP-positive spermatogonia, suggesting that cells in the Aal spermatogonia could revert to a more primitive state.

Regulation of male fertility by X-linked genes.

Infertility is a worldwide reproductive health problem, affecting men and women about equally. Mouse genetic studies demonstrate that more than 200 genes specifically or predominantly regulate fertility. However, few genetic causes of infertility in humans have been identified. Here, we focus on the regulation of male fertility by X-linked, germ cell-specific genes. Previous genomic studies reveal that the mammalian X chromosome is enriched for genes expressed in early spermatogenesis. Recent genetic studies in mice show that X-linked, germ cell-specific genes, such as A-kinase anchor protein 4 (Akap4), nuclear RNA export factor 2 (Nxf2), TBP-associated factor 7l (Taf7l), and testis-expressed gene 11 (Tex11), indeed play important roles in the regulation of male fertility. Moreover, we find that the Taf7l Tex11 double-mutant males exhibit much more severe defects in meiosis than either single mutant, suggesting that these 2 X-linked genes regulate male meiosis synergistically. The X-linked, germ cell-specific genes are particularly attractive in the study of male infertility in humans. Because males are hemizygous for X-linked genes, loss-of-function mutations in the single-copy X-linked genes, unlike in autosomal genes, would not be masked by a normal allele. The genetic studies of X-linked, germ cell-specific genes in mice have laid a foundation for mutational analysis of their human orthologues in infertile men.

The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis.

Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19) has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.

Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis.

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

Meiotic failure in male mice lacking an X-linked factor.

Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes.