Sustained fertility from first-wave follicle oocytes that pause their growth.

Ovulation results from the cyclical recruitment of non-renewing, quiescent oocytes for growth. Therefore, the primordial follicles that are established during development from an oocyte encapsulated by granulosa cells are thought to comprise the lifelong ovarian reserve 1-4. However, using oocyte lineage tracing in mice, we observed that a subset of oocytes recruited for growth in the first juvenile wave remain paused for many months before continuing growth, ovulation, fertilization and development into healthy offspring. This small subset of genetically-labeled fetal oocytes, labeled with Sycp3-CreERT2, is distinguished by earlier entry and slower dynamics of meiotic prophase I. While labeled oocytes were initially found in both primordial follicles and growing follicles of the first wave, they disappeared from primordial follicles by puberty. Unexpectedly, these first-wave labeled growing oocytes persisted throughout reproductive lifespan and contributed to offspring at a steady rate beyond 12 months of age, suggesting that follicles can pause mid-growth for extended periods then successfully resume. These results challenge the conclusion from lineage tracing of granulosa cells that first-wave follicles make a limited contribution to fertility5 and furthermore suggest that growth-paused oocytes comprise a second and previously unrecognized ovarian reserve.

Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants.

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. We previously found that interaction with different somatic niches regulates PGC proliferation along the migration route. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5; these differences include migration-associated actin polymerization machinery and epigenetic reprogramming-associated genes. We furthermore identified changes in signaling with various somatic niches, notably strengthened interactions with hindgut epithelium via non-canonical WNT (ncWNT) in posterior PGCs compared to anterior. Reanalysis of a previously published dataset suggests that ncWNT signaling from the hindgut epithelium to early migratory PGCs is conserved in humans. Trajectory inference methods identified putative differentiation trajectories linking cell states across timepoints and from posterior to anterior in our mouse dataset. At E9.5, we mainly observed differences in cell adhesion and actin cytoskeletal dynamics between E9.5 posterior and anterior migrants. At E10.5, we observed divergent gene expression patterns between putative differentiation trajectories from posterior to anterior including Nodal signaling response genes Lefty1, Lefty2, and Pycr2 and reprogramming factors Dnmt1, Prc1, and Tet1. At E10.5, we experimentally validated anterior migrant-specific Lefty1/2 upregulation via whole-mount immunofluorescence staining for LEFTY1/2 proteins, suggesting that elevated autocrine Nodal signaling accompanies the late stages of PGC migration. Together, this positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency, and epigenetic reprogramming.

Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus.

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.

Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Transcriptional repression upon S phase entry protects genome integrity in pluripotent cells.

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication.

A Roadmap for Three-Dimensional Analysis of the Intact Mouse Ovary.

Recent advances in tissue clearing methodologies have enabled three-dimensional (3D) visualization of the ovary and, consequently, in-depth exploration of the dynamic changes occurring at the single-cell level. Here we describe methods for whole-mount immunofluorescence, clearing, imaging, and analysis of whole ovarian tissue in 3D throughout murine development and aging.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma.

The neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Ovary Development: Insights From a Three-Dimensional Imaging Revolution.

The ovary is an indispensable unit of female reproduction and health. However, the study of ovarian function in mammals is hindered by unique challenges, which include the desynchronized development of oocytes, irregular distribution and vast size discrepancy of follicles, and dynamic tissue remodeling during each hormonal cycle. Overcoming the limitations of traditional histology, recent advances in optical tissue clearing and three-dimensional (3D) visualization offer an advanced platform to explore the architecture of intact organs at a single cell level and reveal new relationships and levels of organization. Here we summarize the development and function of ovarian compartments that have been delineated by conventional two-dimensional (2D) methods and the limits of what can be learned by these approaches. We compare types of optical tissue clearing, 3D analysis technologies, and their application to the mammalian ovary. We discuss how 3D modeling of the ovary has extended our knowledge and propose future directions to unravel ovarian structure toward therapeutic applications for ovarian disease and extending female reproductive lifespan.

Apoptosis in the fetal testis eliminates developmentally defective germ cell clones.

Many germ cells are eliminated during development, long before oogenesis or spermatogenesis. In mouse fetal testes, the majority of germ cell apoptosis coincides with the onset of male differentiation, suggesting coordination of these processes. We studied fetal germ-cell fates and discovered that both apoptosis and differentiation initiate in clonally related clusters. Lineage tracing confirmed that germ cells die as clones independent of intercellular bridges, suggesting that shared intrinsic properties are apoptotic determinants. We identified transcriptional heterogeneity among fetal germ cells that included an apoptosis-susceptible population characterized by failure to differentiate, whereas successful differentiation to prospermatogonia occurred through the expression of epigenetically regulated genes, including LINE1. Our results indicate that the fetal germ-cell fate is based on discrete cell-heritable identities. Elevated DNA methylation in the apoptosis-susceptible subpopulation supports our hypothesis that earlier errors in germ-cell epigenetic reprogramming derail differentiation in cellular progeny, leading to fetal apoptotic selection that ultimately improves the gamete quality.

The UCSF Mouse Inventory Database Application, an Open Source Web App for Sharing Mutant Mice Within a Research Community.

The UCSF Mouse Inventory Database Application is an open-source Web App that provides information about the mutant alleles, transgenes, and inbred strains maintained by investigators at the university and facilitates sharing of these resources within the university community. The Application is designed to promote collaboration, decrease the costs associated with obtaining genetically-modified mice, and increase access to mouse lines that are difficult to obtain. An inventory of the genetically-modified mice on campus and the investigators who maintain them is compiled from records of purchases from external sources, transfers from researchers within and outside the university, and from data provided by users. These data are verified and augmented with relevant information harvested from public databases, and stored in a succinct, searchable database secured on the university network. Here we describe this resource and provide information about how to implement and maintain such a mouse inventory database application at other institutions.

Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

Heterogeneity of primordial germ cells.

Primordial germ cells (PGCs) must complete a complex and dynamic developmental program during embryogenesis to establish the germline. This process is highly conserved and involves a diverse array of tasks required of PGCs, including migration, survival, sex differentiation, and extensive epigenetic reprogramming. A common theme across many organisms is that PGC success is heterogeneous: only a portion of all PGCs complete all these steps while many other PGCs are eliminated from further germline contribution. The differences that distinguish successful PGCs as a population are not well understood. Here, we examine variation that exists in PGCs as they navigate the many stages of this developmental journey. We explore potential sources of PGC heterogeneity and their potential implications in affecting germ cell behaviors. Lastly, we discuss the potential for PGC development to function as a multistage selection process that assesses heterogeneity in PGCs to refine germline quality.

Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells.

Epigenetic resetting in germ cells during development de-represses transposable elements (TEs). piRNAs protect fetal germ cells by targeted mRNA destruction and deposition of repressive epigenetic marks. Here, we provide the first evidence for an active piRNA pathway and TE repression in germ cells of human fetal testis. We identify pre-pachytene piRNAs with features of secondary amplification that map most abundantly to the long interspersed element type 1 (L1) family of TEs. L1-ORF1p expression is heterogeneous in fetal germ cells, peaks at mid-gestation and declines concomitantly with increases in piRNAs, nuclear localization of HIWI2 and an increase in H3K9me3. Surprisingly, the same cells with accumulation of L1-ORF1p display highest levels of HIWI2 and H3K9me3. Conversely, the earliest germ cells with high levels of L1-ORF1p express low levels of the chaperone HSP90α. We propose that a subset of germ cells resists L1 expression, whereas L1-expressing germ cells activate the repression pathway that leads to epigenetic silencing of L1 via H3K9me3.