The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.

Genome-wide recombination is essential for genome stability, evolution, and speciation. Mouse Tex11, an X-linked meiosis-specific gene, promotes meiotic recombination and chromosomal synapsis. Here, we report that TEX11 is mutated in infertile men with non-obstructive azoospermia and that an analogous mutation in the mouse impairs meiosis. Genetic screening of a large cohort of idiopathic infertile men reveals that TEX11 mutations, including frameshift and splicing acceptor site mutations, cause infertility in 1% of azoospermic men. Functional evaluation of three analogous human TEX11 missense mutations in transgenic mouse models identified one mutation (V748A) as a potential infertility allele and found two mutations non-causative. In the mouse model, an intronless autosomal Tex11 transgene functionally substitutes for the X-linked Tex11 gene, providing genetic evidence for the X-to-autosomal retrotransposition evolution phenomenon. Furthermore, we find that TEX11 protein levels modulate genome-wide recombination rates in both sexes. These studies indicate that TEX11 alleles affecting expression level or substituting single amino acids may contribute to variations in recombination rates between sexes and among individuals in humans.

Although the mammalian X and Y chromosomes evolved from a single pair of autosomes, they are highly differentiated: the Y chromosome is dramatically smaller than the X and has lost most of its genes. The surviving genes are a specialized set with extraordinary evolutionary longevity. Most mammalian lineages have experienced delayed, or relatively recent, loss of at least one conserved Y-linked gene. An extreme example of this phenomenon is in the Japanese spiny rat, where the Y chromosome has disappeared altogether. In this species, many Y-linked genes were rescued by transposition to new genomic locations, but until our work presented here, this has been considered an isolated case.

Amplicons--large, nearly identical repeats in direct or inverted orientation--are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms.

In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.

Mammalian spermatogenesis is an elaborately organized differentiation process, starting with diploid spermatogonia, which include germ-line stem cells, and ending with haploid spermatozoa. The process involves four pivotal transitions occurring in physical proximity: spermatogonial differentiation, meiotic initiation, initiation of spermatid elongation, and release of spermatozoa. We report how the four transitions are coordinated in mice. Two premeiotic transitions, spermatogonial differentiation and meiotic initiation, were known to be coregulated by an extrinsic signal, retinoic acid (RA). Our chemical manipulations of RA levels in mouse testes now reveal that RA also regulates the two postmeiotic transitions: initiation of spermatid elongation and spermatozoa release. We measured RA concentrations and found that they changed periodically, as also reflected in the expression patterns of an RA-responsive gene, STRA8; RA levels were low before the four transitions, increased when the transitions occurred, and remained elevated thereafter. We found that pachytene spermatocytes, which express an RA-synthesizing enzyme, Aldh1a2, contribute directly and significantly to RA production in testes. Indeed, chemical and genetic depletion of pachytene spermatocytes revealed that RA from pachytene spermatocytes was required for the two postmeiotic transitions, but not for the two premeiotic transitions. We conclude that the premeiotic transitions are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate the two postmeiotic transitions. In combination, these elements underpin the spatiotemporal coordination of spermatogenesis and ensure its prodigious output in adult males.

Germ cells undergo many developmental transitions before ultimately becoming either eggs or sperm, and during embryonic development these transitions include epigenetic reprogramming, quiescence, and meiosis. To begin understanding the transcriptional regulation underlying these complex processes, we examined the spatial and temporal expression of TAF4b, a variant TFIID subunit required for fertility, during embryonic germ cell development. By analyzing published datasets and using our own experimental system to validate these expression studies, we determined that both Taf4b mRNA and protein are highly germ cell-enriched and that Taf4b mRNA levels dramatically increase from embryonic day 12.5-18.5. Surprisingly, additional mRNAs encoding other TFIID subunits are coordinately upregulated through this time course, including Taf7l and Taf9b. The expression of several of these germ cell-enriched TFIID genes is dependent upon Dazl and/or Stra8, known regulators of germ cell development and meiosis. Together, these data suggest that germ cells employ a highly specialized and dynamic form of TFIID to drive the transcriptional programs that underlie mammalian germ cell development.

Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

The special cell cycle known as meiosis transforms diploid germ cells into haploid gametes. In mammalian testes, diploid spermatogenic cells become competent to transition from mitosis to meiosis in response to retinoic acid. In mice, previous studies revealed that MEIOC, alongside binding partners YTHDC2 and RBM46, represses mitotic genes and promotes robust meiotic gene expression in spermatogenic cells that have already initiated meiosis. Here, we molecularly dissect MEIOC-dependent regulation in mouse spermatogenic cells and find that MEIOC actually shapes the transcriptome much earlier, even before meiotic initiation. MEIOC, acting with YTHDC2 and RBM46, destabilizes mRNA targets, including transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the STRA8-MEIOSIN transcriptional regulator, which is required for the meiotic G1/S cell cycle transition and meiotic gene expression. We conclude that in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of mitotic spermatogonia to transit from mitosis to meiosis.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes.

It is widely believed that at least three nonoverlapping regions of the human Y chromosome-AZFa, AZFb, and AZFc ("azoospermia factors" a, b, and c)-are essential for normal spermatogenesis. These intervals are defined by interstitial Y-chromosome deletions that impair or extinguish spermatogenesis. Deletion breakpoints, mechanisms, and lengths, as well as inventories of affected genes, have been elucidated for deletions of AZFa and of AZFc but not for deletions of AZFb or of AZFb plus AZFc. We studied three deletions of AZFb and eight deletions of AZFb plus AZFc, as assayed by the STSs defining these intervals. Guided by Y-chromosome sequence, we localized breakpoints precisely and were able to sequence nine of the deletion junctions. Homologous recombination can explain seven of these deletions but not the remaining two. This fact and our discovery of breakpoint hotspots suggest that factors in addition to homology underlie these deletions. The deletions previously thought to define AZFb were found to extend from palindrome P5 to the proximal arm of palindrome P1, 1.5 Mb within AZFc. Thus, they do not define a genomic region separate from AZFc. We also found that the deletions of AZFb plus AZFc, as assayed by standard STSs heretofore available, in fact extend from P5 to the distal arm of P1 and spare distal AZFc. Both classes of deletions are massive: P5/proximal-P1 deletions encompass up to 6.2 Mb and remove 32 genes and transcripts; P5/distal-P1 deletions encompass up to 7.7 Mb and remove 42 genes and transcripts. To our knowledge, these are the largest of all human interstitial deletions for which deletion junctions and complete intervening sequence are available. The restriction of the associated phenotype to spermatogenic failure indicates the remarkable functional specialization of the affected regions of the Y chromosome.

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.

Oogenesis is the process by which ovarian germ cells undertake meiosis and differentiate to become eggs. In mice, Stra8 is required for the chromosomal events of meiosis to occur, but its role in differentiation remains unknown. Here we report Stra8-deficient ovarian germ cells that grow and differentiate into oocyte-like cells that synthesize zonae pellucidae, organize surrounding somatic cells into follicles, are ovulated in response to hormonal stimulation, undergo asymmetric cell division to produce a polar body and cleave to form two-cell embryos upon fertilization. These events occur without premeiotic chromosomal replication, sister chromatid cohesion, synapsis or recombination. Thus, oocyte growth and differentiation are genetically dissociable from the chromosomal events of meiosis. These findings open to study the independent contributions of meiosis and oocyte differentiation to the making of a functional egg.

Amplicons-large, highly identical segmental duplications-are a prominent feature of mammalian Y chromosomes. Although they encode genes essential for fertility, these amplicons differ vastly between species, and little is known about the selective constraints acting on them. Here, we develop computational tools to detect amplicon copy number with unprecedented accuracy from high-throughput sequencing data. We find that one-sixth (16.9%) of 1,216 males from the 1000 Genomes Project have at least one deleted or duplicated amplicon. However, each amplicon's reference copy number is scrupulously maintained among divergent branches of the Y chromosome phylogeny, including the ancient branch A00, indicating that the reference copy number is ancestral to all modern human Y chromosomes. Using phylogenetic analyses and simulations, we demonstrate that this pattern of variation is incompatible with neutral evolution and instead displays hallmarks of mutation-selection balance. We also observe cases of amplicon rescue, in which deleted amplicons are restored through subsequent duplications. These results indicate that, contrary to the lack of constraint suggested by the differences between species, natural selection has suppressed amplicon copy number variation in diverse human lineages.

No abstract available

The human Y chromosome began to evolve from an autosome hundreds of millions of years ago, acquiring a sex-determining function and undergoing a series of inversions that suppressed crossing over with the X chromosome. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. Prevailing theories hold that Y chromosomes evolve by gene loss, the pace of which slows over time, eventually leading to a paucity of genes, and stasis. These theories have been buttressed by partial sequence data from newly emergent plant and animal Y chromosomes, but they have not been tested in older, highly evolved Y chromosomes such as that of humans. Here we finished sequencing of the male-specific region of the Y chromosome (MSY) in our closest living relative, the chimpanzee, achieving levels of accuracy and completion previously reached for the human MSY. By comparing the MSYs of the two species we show that they differ radically in sequence structure and gene content, indicating rapid evolution during the past 6 million years. The chimpanzee MSY contains twice as many massive palindromes as the human MSY, yet it has lost large fractions of the MSY protein-coding genes and gene families present in the last common ancestor. We suggest that the extraordinary divergence of the chimpanzee and human MSYs was driven by four synergistic factors: the prominent role of the MSY in sperm production, 'genetic hitchhiking' effects in the absence of meiotic crossing over, frequent ectopic recombination within the MSY, and species differences in mating behaviour. Although genetic decay may be the principal dynamic in the evolution of newly emergent Y chromosomes, wholesale renovation is the paramount theme in the continuing evolution of chimpanzee, human and perhaps other older MSYs.

Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3' UTRs of ~2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3' UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors-ZFX and ZFY-encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonad.

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning.