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In vitro gametogenesis (IVG), the reconstitution of germ cell development in vitro, is an emerging stem cell-based technology with profound implications for reproductive science. Despite researchers' long-term goals for future clinical applications, little is currently known about the views of IVG held by the stakeholders potentially most affected by its introduction in humans. We conducted focus groups and interviews with 80 individuals with lived experience of infertility and/or LGBTQ+ family formation in the US, two intersecting groups of potential IVG users. Respondents expressed hope that IVG would lead to higher reproductive success than current assisted reproductive technology (ART), alleviate suffering associated with ART use, and promote greater social inclusion, while expressing concerns predominantly framed in terms of equity and safety. These findings underscore the importance of sustained engagement with stakeholders with relevant experience to anticipate the implications of IVG for research and clinical translation.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.

Metabolism participates in the control of stem cell function and subsequent maintenance of tissue homeostasis. How this is achieved in the context of adult stem cell niches in coordination with other local and intrinsic signaling cues is not completely understood. The Target of Rapamycin (TOR) pathway is a master regulator of metabolism and plays essential roles in stem cell maintenance and differentiation. In the Drosophila male germline, mTORC1 is active in germline stem cells (GSCs) and early germ cells. Targeted RNAi-mediated downregulation of mTor in early germ cells causes a block and/or a delay in differentiation, resulting in an accumulation of germ cells with GSC-like features. These early germ cells also contain unusually large and dysfunctional autolysosomes. In addition, downregulation of mTor in adult male GSCs and early germ cells causes non-autonomous activation of mTORC1 in neighboring cyst cells, which correlates with a disruption in the coordination of germline and somatic differentiation. Our study identifies a previously uncharacterized role of the TOR pathway in regulating male germline differentiation.

Formation of either an ovary or a testis during human embryonic life is one of the most important sex-specific events leading to the emergence of secondary sexual characteristics and sex assignment of babies at birth. Our study focused on the sex-specific and sex-indifferent characteristics of the prenatal ovarian stromal cells, cortical cords, and germline, with the discovery that the ovarian mesenchymal cells of the stroma are transcriptionally indistinguishable from the mesenchymal cells of the testicular interstitium. We found that first-wave pre-granulosa cells emerge at week 7 from early supporting gonadal cells with stromal identity and are spatially defined by KRT19 levels. We also identified rare transient state f0 spermatogonia cells within the ovarian cords between weeks 10 and 16. Taken together, our work illustrates a unique plasticity of the embryonic ovary during human development.

Objective To investigate the effect of endoplasmic reticulum associated protein VAPA on spermiogenesis in mice. Methods A conditional knockout mouse model with specific Vapa gene deletion in germ cells (Vapa CKO) was constructed using the loxP-cre strategy. Testes collected from wild-type and Vapa CKO adult male mice were weighed for statistical analysis. HE staining was performed to analyze the histological morphology of the seminiferous epithelium. Immunofluorescence staining and transmission electron microscopy were used to determine acrosome biogenesis in wild-type and Vapa CKO adult male mice. Results The weight of testes from Vapa CKO adult male mice exhibited a significant decrease (P <0.000 1). Specifically, spermatogenesis was arrested at round spermatids in Vapa CKO mice because round spermatids were unable to generate acrosome granule. Conclusion VAPA is involved in mouse spermiogenesis and plays an important role in acrosome biogenesis. [ABSTRACT FROM AUTHOR]

Germline mosaicisms could be inherited to offspring, which considered as "de novo" in most cases. Paternal germline MECP2 mosaicism has been reported in fathers of girls with Rett syndrome (RTT) previously. For further study, we focused on MECP2 germline mosaicism in males, not only RTT fathers. Thirty-two fathers of RTT girls with MECP2 pathogenic mutations and twenty-five healthy adult males without history and family history of RTT or other genetic disorders were recruited. Sperm samples were collected and ten MECP2 hotspot mutations were detected by micro-droplet digital PCR (mDDPCR). And routine semen test was performed at the same time if the sample was sufficient. Additionally, blood samples were also detected for those with sperm MECP2 mosaicisms. Nine fathers with RTT daughters (28.1%, 9/32) were found to have MECP2 mosaicism in their sperm samples, with the mutant allele fractions (MAFs) ranging from 0.05% to 7.55%. Only one father with MECP2 c.806delG germline mosaicism (MAF 7.55%) was found to have mosaicism in the blood sample, with the MAF was 0.28%. In the group of healthy adult males, MECP2 mosaicism was found in 7 sperm samples (28.0%, 7/25), with the MAFs ranging from 0.05% to 0.18%. None of the healthy adult males with MECP2 germline mosaicisms were found with MECP2 mosaicism in blood samples. There were no statistical differences in age, or the incidence of asthenospermia between fathers with RTT daughters and healthy adult males with MECP2 germline mosaicisms. Additionally, there was no linear correlation between MAFs of MECP2 mosaicisms and the age of males with germline MECP2 mosaicisms. Germline MECP2 mosaicism could be found not only in fathers with RTT daughters but also in healthy adult males without family history of RTT. As germline mosaic mutations may be passed on to offspring which commonly known as "de novo", more attention should be paid to germline mosaicism, especially in families with a proband diagnosed with genetic disorders.

Pluripotent stem cells (PSC) can be stabilized in vitro from pre-implantation stage embryos (embryonic stem cells, ESC) or by reprogramming adult somatic cells (induced pluripotent stem cells, iPSC). The last decade has seen significant advances in the livestock PSC field, particularly the development of robust methods for long-term culture of PSC from several livestock species. Along with this, considerable progress has been made in understanding the states of cellular pluripotency and what they mean for cell differentiation capacity, and significant efforts are ongoing to dissect the critical signaling pathways required for the maintenance of PSC in different species and distinct states of pluripotency. Among the cell types that can be generated from PSC, the germline holds special importance as they are the genetic link between generations; and devising methods to enable in vitro gametogenesis (IVG) and produce viable gametes could revolutionize animal agriculture, wildlife conservation, and human assisted reproduction alike. Within the last decade, many pivotal studies about IVG were published using rodent models, filling some critical knowledge gaps in the field. Most importantly, the entire female reproductive cycle was reproduced in vitro from mouse ESC. Although complete male gametogenesis in vitro has not yet been reported, significant advances were made showing the capacity of germline stem cell-like cells to generate healthy offspring. In this review, we provide an overview of PSC and advances in the establishment of livestock PSC; we present the breakthroughs made in rodents regarding IVG and the current progress towards livestock IVG, including the importance of a detailed understanding of fetal germline development. Finally, we discuss some key advances that will be critical to enable this technology at scale. Given the potential impact of IVG for animal agriculture, major efforts will likely continue to be employed by research institutions and industry towards the development of methods to achieve efficient generation of gametes in vitro.

We present a case of SCALP syndrome, which was diagnosed in a male infant with the characteristic findings of sebaceous nevi, central nervous system malformations, aplasia cutis congenita, limbal dermoid, and giant congenital melanocytic nevi, or pigmented nevi. We identified a germline compound heterozygous DOCK6 mutation and a somatic mosaic NRAS Q61R mutation in the giant congenital melanocytic nevus. This report will increase clinician awareness of SCALP syndrome and augment the literature in characterizing this rare syndrome, including its genetic background.

People are widely exposed to polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP). Prior studies showed that prenatal exposure to BaP depletes germ cells in ovaries, causing earlier onset of ovarian senescence post-natally; developing testes were affected at higher doses than ovaries. Our primary objective was to determine if prenatal BaP exposure results in transgenerational effects on ovaries and testes. We orally dosed pregnant germ cell-specific EGFP-expressing mice (F0) with 0.033, 0.2, or 2 mg/kg-day BaP or vehicle from embryonic day (E) 6.5-11.5 (F1 offspring) or E6.5-15.5 (F2 and F3). Ovarian germ cells at E13.5 and follicle numbers at postnatal day 21 were significantly decreased in F3 females at all doses of BaP; testicular germ cell numbers were not affected. E13.5 germ cell RNA-sequencing revealed significantly increased expression of male-specific genes in female germ cells across generations and BaP doses. Next, we compared the ovarian effects of 2 mg/kg-day BaP dosing to wild type C57BL/6J F0 dams from E6.5-11.5 or E12.5-17.5. We observed no effects on F3 ovarian follicle numbers with either of the shorter dosing windows. Our results demonstrate that F0 BaP exposure from E6.5-15.5 decreased the number of and partially disrupted transcriptomic sexual identity of female germ cells transgenerationally.

Unlike PIWI-interacting RNA (piRNA) in other species that mostly target transposable elements (TEs), >80% of piRNAs in adult mammalian testes lack obvious targets. However, mammalian piRNA sequences and piRNA-producing loci evolve more rapidly than the rest of the genome for unknown reasons. Here, through comparative studies of chickens, ducks, mice, and humans, as well as long-read nanopore sequencing on diverse chicken breeds, we find that piRNA loci across amniotes experience: (1) a high local mutation rate of structural variations (SVs, mutations ≥ 50 bp in size); (2) positive selection to suppress young and actively mobilizing TEs commencing at the pachytene stage of meiosis during germ cell development; and (3) negative selection to purge deleterious SV hotspots. Our results indicate that genetic instability at pachytene piRNA loci, while producing certain pathogenic SVs, also protects genome integrity against TE mobilization by driving the formation of rapid-evolving piRNA sequences.

Prostate cancer is one of the most heritable human cancers. Genome-wide association studies have identified at least 185 prostate cancer germline risk alleles, most noncoding. We used integrative three-dimensional (3D) spatial genomics to identify the chromatin interaction targets of 45 prostate cancer risk alleles, 31 of which were associated with the transcriptional regulation of target genes in 565 localized prostate tumors. To supplement these 31, we verified transcriptional targets for 56 additional risk alleles using linear proximity and linkage disequilibrium analysis in localized prostate tumors. Some individual risk alleles influenced multiple target genes; others specifically influenced only distal genes while leaving proximal ones unaffected. Several risk alleles exhibited widespread germline-somatic interactions in transcriptional regulation, having different effects in tumors with loss of PTEN or RB1 relative to those without. These data clarify functional prostate cancer risk alleles in large linkage blocks and outline a strategy to model multidimensional transcriptional regulation.SignificanceMany prostate cancer germline risk alleles are enriched in the noncoding regions of the genome and are hypothesized to regulate transcription. We present a 3D genomics framework to unravel risk SNP function and describe the widespread germline-somatic interplay in transcription control. This article is highlighted in the In This Issue feature, p. 2711.

Radiofrequency electromagnetic radiation (RF-EMR) from various sources may impact health due to the generation of frequency bands. Broad pulses emitted within frequency bands can be absorbed by cells, influencing their function. Numerous laboratory studies have demonstrated that mobile phones--generally the most widely used devices--can have harmful effects on sex cells, such as sperm and oocytes, by producing RF-EMR. Moreover, some research has indicated that RF-EMR generated by mobile phones can influence sperm parameters, including motility, morphology, viability, and (most critically) DNA structure. Consequently, RF-EMR can disrupt both sperm function and fertilization. However, other studies have reported that exposure of spermatozoa to RF-EMR does not affect the functional parameters or genetic structure of sperm. These conflicting results likely stem from differences among studies in the duration and exposure distance, as well as the species of animal used. This report was undertaken to review the existing research discussing the effects of RF-EMR on the DNA integrity of mammalian spermatozoa. [ABSTRACT FROM AUTHOR]

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.

A male mutation bias is observed across vertebrates, and, where data are available, this bias is accompanied by increased per-generation mutation rates with parental age. While continuing mitotic cell division in the male germline post puberty has been proposed as the major cellular mechanism underlying both patterns, little direct evidence for this role has been found. Understanding the evolution of the per-generation mutation rate among species requires that we identify the molecular mechanisms that change between species. Here, we study the per-generation mutation rate in an extended pedigree of the brown (grizzly) bear, Ursus arctos horribilis. Brown bears hibernate for one-third of the year, a period during which spermatogenesis slows or stops altogether. The reduction of spermatogenesis is predicted to lessen the male mutation bias and to lower the per-generation mutation rate in this species. However, using whole-genome sequencing, we find that both male bias and per-generation mutation rates are highly similar to that expected for a non-hibernating species. We also carry out a phylogenetic comparison of substitution rates along the lineage leading to brown bear and panda (a non-hibernating species) and find no slowing of the substitution rate in the hibernator. Our results contribute to accumulating evidence that suggests that male germline cell division is not the major determinant of mutation rates and mutation biases. The results also provide a quantitative basis for improved estimates of the timing of carnivore evolution.

Germline testing is becoming increasingly relevant in prostate cancer (PCa) screening, prognosis, and management. A subset of patients with PCa harbor pathogenic/likely pathogenic variants (P/LPVs) in genes mediating DNA-repair processes, and these P/LPVs have implications for cancer screening, treatment, and cascade testing. As a result, it is recommended that all men with high-risk localized and metastatic PCa undergo routine germline testing. As more PCa patients undergo germline testing, it is important that clinicians and genetics experts recognize current disparities in germline testing rates among racial/ethnic minorities in the United States. The reasons for these disparities are multiple and require similarly manifold consideration to close the germline testing gap and reduce inequities in PCa screening, management, and treatment.

Primary pulmonary choriocarcinoma is a highly aggressive germ cell neoplasm and an extremely rare, especially in males. It is characterized by a poor response to therapy and shortened survival times. We present the case of primary pulmonary choriocarcinoma in a 46‐year‐old male. The patient was referred to our institute with cough, worsening dyspnea and hemoptysis. The contrast‐enhanced chest computed tomography revealed an avid enhanced 15 × 14 cm sized nodular lesion, in the left lower lung, which invaded into the diaphragm. After the embolization of the intercostal arteries, the tumour was resected successfully. However, the patient had died suddenly on the 28th day after the surgery. Autopsy was conducted and revealed that his cause of the death was the tumour emboli in the right coronary artery. [ABSTRACT FROM AUTHOR]

Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

Background and purposeThe purpose of this study was to determine whether single nucleotide polymorphisms disrupting microRNA targets (mirSNPs) can serve as predictive biomarkers for toxicity after radiotherapy for prostate cancer and whether these may be differentially predictive depending on radiation fractionation.Materials and methodsWe identified 201 men treated with two forms of definitive radiotherapy for prostate cancer at two institutions: 108 men received conventionally-fractionated radiotherapy (CF-RT) and 93 received stereotactic body radiotherapy (SBRT). Germline DNA was evaluated for the presence of functional mirSNPs. Random forest, boosted trees and elastic net models were developed to predict late grade ≥2 GU toxicity by the RTOG scale.ResultsThe crude incidence of late grade ≥2 GU toxicity was 16% after CF-RT and 15% after SBRT. An elastic net model based on 22 mirSNPs differentiated CF-RT patients at high risk (71.5%) versus low risk (7.5%) for toxicity, with an area under the curve (AUC) values of 0.76-0.81. An elastic net model based on 32 mirSNPs differentiated SBRT patients at high risk (64.7%) versus low risk (3.9%) for toxicity, with an area under the curve (AUC) values of 0.81-0.87. These models were specific to treatment type delivered. Prospective studies are warranted to further validate these results.ConclusionPredictive models using germline mirSNPs have high accuracy for predicting late grade ≥2 GU toxicity after either CF-RT or SBRT, and are unique for each treatment, suggesting that germline predictors of late radiation sensitivity are fractionation-dependent. Prospective studies are warranted to further validate these results.

Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNosCd-1 drive (nos-Cas9) in Anopheles gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly coexpressed with endogenous genes in stem and premeiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct colocalization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.

When two species meet in secondary contact, the production of low fitness hybrids may be prevented by the adaptive evolution of increased prezygotic isolation, a process known as reinforcement. Theoretical challenges to the evolution of reinforcement are generally cast as a coordination problem, i.e., "how can statistical associations between traits and preferences be maintained in the face of recombination?" However, the evolution of reinforcement also poses a potential conflict between mates. For example, the opportunity costs to hybridization may differ between the sexes or species. This is particularly likely for reinforcement based on postmating prezygotic (PMPZ) incompatibilities, as the ability to fertilize both conspecific and heterospecific eggs is beneficial to male gametes, but heterospecific mating may incur a cost for female gametes. We develop a population genetic model of interspecific conflict over reinforcement inspired by "gametophytic factors", which act as PMPZ barriers among Zea mays subspecies. We demonstrate that this conflict results in the transient evolution of reinforcement-after females adaptively evolve to reject gametes lacking a signal common in conspecific gametes, this gamete signal adaptively introgresses into the other population. Ultimately, the male gamete signal fixes in both species, and isolation returns to pre-reinforcement levels. We interpret geographic patterns of isolation among Z. mays subspecies considering these findings and suggest when and how this conflict can be resolved. Our results suggest that sexual conflict over fertilization may pose an understudied obstacle to the evolution of reinforcement.

CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

Sertoli cells (SCs) support and nourish germ cells (GCs) through their crosstalk during spermatogenesis. However, the underlying epigenetic mechanism that ensures SCs' functions in this process remains unclear. Here, we report that UHRF1, a critical epigenetic regulator, is mainly expressed in human and mouse pre-mature SCs, and is essential for establishing Sertoli-Germ cell crosstalk. SC-specific UHRF1 knockout mice exhibit complete sterility with Sertoli cell (SC) proliferation and differentiation aberrance, blood-testis barrier (BTB) disruption, and immature germ cell (GC) sloughing. RNA sequencing and Whole Genome Bisulfite Sequencing (WGBS) revealed that many extracellular matrix (ECM)-related genes (e.g., Timp1, Trf, and Spp1) appeared upregulated with the DNA hypomethylation status in UHRF1-deficient SCs. Strikingly, overexpression of Timp1, Trf, and Spp1 in SCs in vitro and in vivo could phenocopy the SC-specific UHRF1-deficient mice. Our data demonstrated that UHRF1 regulates the transcriptional program of ECM-related genes in SCs and establishes SC-GC crosstalk.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

Spermatogonial stem cells (SSCs) are essential for male fertility. Here, we report that mouse SSC generation is driven by a transcription factor (TF) cascade controlled by the homeobox protein, RHOX10, which acts by driving the differentiation of SSC precursors called pro-spermatogonia (ProSG). We identify genes regulated by RHOX10 in ProSG in vivo and define direct RHOX10-target genes using several approaches, including a rapid temporal induction assay: iSLAMseq. Together, these approaches identify temporal waves of RHOX10 direct targets, as well as RHOX10 secondary-target genes. Many of the RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay, we find that RHOX10 promotes mouse ProSG differentiation through a conserved transcriptional cascade involving the key germ-cell TFs DMRT1 and ZBTB16. Our study gives important insights into germ cell development and provides a blueprint for how to define TF cascades.

Transposable elements (TEs) are mobile sequences that engender widespread mutations and thus are a major hazard that must be silenced. The most abundant active class of TEs in mammalian genomes is long interspersed element class 1 (LINE1). Here, we report that LINE1 transposition is suppressed in the male germline by transcription factors encoded by a rapidly evolving X-linked homeobox gene cluster. LINE1 transposition is repressed by many members of this RHOX transcription factor family, including those with different patterns of expression during spermatogenesis. One family member-RHOX10-suppresses LINE1 transposition during fetal development in vivo when the germline would otherwise be susceptible to LINE1 activation because of epigenetic reprogramming. We provide evidence that RHOX10 suppresses LINE transposition by inducing Piwil2, which encodes a key component in the Piwi-interacting RNA pathway that protects against TEs. The ability of RHOX transcription factors to suppress LINE1 is conserved in humans but is lost in RHOXF2 mutants from several infertile human patients, raising the possibility that loss of RHOXF2 causes human infertility by allowing uncontrolled LINE1 expression in the germline. Together, our results support a model in which the Rhox gene cluster is in an evolutionary arms race with TEs, resulting in expansion of the Rhox gene cluster to suppress TEs in different biological contexts.

Each human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. Whereas the rate and patterns of point mutations in the human germline are now well understood, far less is known about the frequency and features that impact de novo structural variants (dnSVs). We report a family-based study of germline mutations among 9,599 human genomes from 33 multigenerational CEPH-Utah families and 2,384 families from the Simons Foundation Autism Research Initiative. We find that de novo structural mutations detected by alignment-based, short-read WGS occur at an overall rate of at least 0.160 events per genome in unaffected individuals, and we observe a significantly higher rate (0.206 per genome) in ASD-affected individuals. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and we predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction, we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations and that these mechanisms most likely have markedly different rates and selective pressures than those leading to point mutations.

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.

Germline genetic testing is now routinely recommended for patients with prostate cancer (PCa) because of expanded guidelines and options for targeted treatments. However, integrating genetic testing into oncology and urology clinical workflows remains a challenge because of the increased number of patients with PCa requiring testing and the limited access to genetics providers. This suggests a critical unmet need for genetic services outside of historical models. This review addresses current guidelines, considerations, and challenges for PCa genetic testing and offers a practical guide for genetic counseling and testing delivery, with solutions to help address potential barriers and challenges for both providers and patients. As genetic and genomic testing become integral to PCa care, developing standardized systems for implementation in the clinic is essential for delivering precision oncology to patients with PCa and realizing the full scope and impact of genetic testing.

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.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)-the master regulator of XCI-which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.

First reported in the 1800s, germ cell granules are small nonmembrane bound RNA‐rich regions of the cytoplasm. These sites of critical RNA processing and storage in the male germ cell are essential for proper differentiation and development and are present in a wide range of species from Caenorhabditis elegans through mammals. Initially characterized by light and electron microscopy, more modern techniques such as immunofluorescence and genetic models have played a major role in expanding our understanding of the composition of these structures. While these methods have given light to potential granule functions, much work remains to be done. The current expansion of imaging technologies and omics‐scale analyses to germ cell granule research will drive the field forward considerably. Many of these methods, both current and upcoming, have considerable caveats and limitations that necessitate a holistic approach to the study of germ granules. By combining and balancing different techniques, the field is poised to elucidate the nature of these critical structures. [ABSTRACT FROM AUTHOR]

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.

IntroductionGermline variants in androgen metabolism genes may influence clinical response to androgen deprivation therapy (ADT) in advanced prostate cancer. We sought to investigate the prognostic significance of germline variants in androgen metabolism genes with respect to overall survival (OS) after ADT, and to associate germline variants with tumor genomic features.MethodsGermline and somatic whole-genome sequencing (WGS) data were evaluated in a cohort of 101 men with metastatic castration-resistant prostate cancer (mCRPC). Survival analyses were performed to identify polymorphisms associated with impaired OS after primary ADT. Germline variants found to be prognostic of OS were associated with tumor somatic DNA-sequence alterations based on WGS performed on paired metastasis biopsies from the same 101 patients. Gene set enrichment analysis was performed based on tumor RNA-sequencing data to identify genomic pathways differentially expressed in patients with germline variants.ResultsA comprehensive literature review identified 17 candidate polymorphisms in nine androgen metabolism genes that have been previously shown to have an association with response to ADT in prostate cancer. Of these, the variant rs1856888 allele located 13 kb upstream of HSD3B1 was found to be significantly associated with impaired OS (P = 0.029). Variant rs1856888 was commonly co-inherited with the well-characterized HSD3B1(1245A>C) polymorphism, and there was a trend toward shorter median OS in patients with HSD3B1(1245A>C) compared with homozygous wild-type patients (P = 0.052). While HSD3B1 germline variants were not associated with common somatic tumor DNA alterations, they were associated with increased tumor expression of cell proliferation and cell cycle genes.ConclusionsThis study presents a comprehensive assessment of germline variants in androgen metabolism genes and highlights HSD3B1 polymorphisms as prognostic of OS after ADT and associated with an aggressive gene expression tumor profile in mCRPC.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

The piRNA pathway protects germline genomes from selfish genetic elements (e.g. transposons) through their transcript cleavage in the cytoplasm and/or their transcriptional silencing in the nucleus. Here, we describe a mechanism by which the nuclear and cytoplasmic arms of the piRNA pathway are linked. We find that during mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of the piRNA pathway. At the end of mitosis, Piwi leaves nuage to return to the nucleus. Dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin, Klp10A. Depletion of klp10A delays the return of Piwi to the nucleus and affects piRNA production, suggesting the role of nuclear-cytoplasmic communication in piRNA biogenesis. We propose that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway may play a previously unappreciated role in piRNA regulation.

Although typically upregulated upon cellular stress, autophagy can also be utilized under homeostatic conditions as a quality control mechanism or in response to developmental cues. Here, we report that autophagy is required for the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis. Disruption of autophagy in CySCs and early cyst cells (CCs) by the depletion of autophagy-related (Atg) genes reduced early CC numbers and affected CC function, resembling decreased epidermal growth factor receptor (EGFR) signaling. Indeed, our data indicate that EGFR acts to stimulate autophagy to preserve early CC function, whereas target of rapamycin (TOR) negatively regulates autophagy in the differentiating CCs. Finally, we show that the EGFR-mediated stimulation of autophagy regulates lipid levels in CySCs and CCs. These results demonstrate a key role for autophagy in regulating somatic stem cell behavior and tissue homeostasis by integrating cues from both the EGFR and TOR signaling pathways to control lipid metabolism.

Heteroplasmy-the presence of multiple mitochondrial DNA (mtDNA) haplotypes in an individual-can lead to numerous mitochondrial diseases. The presentation of such diseases depends on the frequency of the heteroplasmic variant in tissues, which, in turn, depends on the dynamics of mtDNA transmissions during germline and somatic development. Thus, understanding and predicting these dynamics between generations and within individuals is medically relevant. Here, we study patterns of heteroplasmy in 2 tissues from each of 345 humans in 96 multigenerational families, each with, at least, 2 siblings (a total of 249 mother-child transmissions). This experimental design has allowed us to estimate the timing of mtDNA mutations, drift, and selection with unprecedented precision. Our results are remarkably concordant between 2 complementary population-genetic approaches. We find evidence for a severe germline bottleneck (7-10 mtDNA segregating units) that occurs independently in different oocyte lineages from the same mother, while somatic bottlenecks are less severe. We demonstrate that divergence between mother and offspring increases with the mother's age at childbirth, likely due to continued drift of heteroplasmy frequencies in oocytes under meiotic arrest. We show that this period is also accompanied by mutation accumulation leading to more de novo mutations in children born to older mothers. We show that heteroplasmic variants at intermediate frequencies can segregate for many generations in the human population, despite the strong germline bottleneck. We show that selection acts during germline development to keep the frequency of putatively deleterious variants from rising. Our findings have important applications for clinical genetics and genetic counseling.

Immune-checkpoint inhibition (ICI) treatments improve outcomes for metastatic melanoma; however, > 60% of treated patients do not respond to ICI. Current biomarkers do not reliably explain ICI resistance. Given the link between ICI and autoimmunity, we investigated if genetic susceptibility to autoimmunity modulates ICI efficacy. In 436 patients with metastatic melanoma receiving single line ICI or combination treatment, we tested 25 SNPs, associated with > 2 autoimmune diseases in recent genome-wide association studies, for modulation of ICI efficacy. We found that rs17388568-a risk variant for allergy, colitis and type 1 diabetes-was associated with increased anti-PD-1 response, with significance surpassing multiple testing adjustments (OR 0.26; 95% CI 0.12-0.53; p = 0.0002). This variant maps to a locus of established immune-related genes: IL2 and IL21. Our study provides first evidence that autoimmune genetic susceptibility may modulate ICI efficacy, suggesting that systematic testing of autoimmune risk loci could reveal personalized biomarkers of ICI response.

Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility.

Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.

The chemotherapeutic drug 5-fluorouracil (5FU) is frequently used to treat a wide range of solid malignant tumors, such as colorectal, pancreatic, gastric, breast, and head and neck cancers. Its antitumoral effects are achieved by interfering with the synthesis of RNA and DNA and by inhibiting thymidylate synthase in both malignant and non-malignant cells. Therefore, it can be responsible for severe toxicities in crucial body organs, including heart, liver, kidney, and reproductive system. Given the fact that 5FU-induced reproductive toxicity may limit the clinical application of this drug, in this study, we aimed to discuss the main locations and mechanisms of the 5FU-induced reproductive toxicity. Initially, we discussed the impact of 5FU on the male reproductive system, which leads to damage of the seminiferous epithelial cells and the development of vacuoles in Sertoli cells. Although no noticeable changes occur at the histopathological level, there is a decrease in the weight of the prostate. Additionally, 5FU causes significant abnormalities in spermatogenesis, including germ cell shedding, spermatid halo formation, polynucleated giant cells, and decreased sperm count. Finally, in females, 5FU-induced reproductive toxicity is characterized by the presence of atretic secondary and antral follicles with reduced numbers of growing follicles, ovarian weight, and maturity impairment. [ABSTRACT FROM AUTHOR]

Epigenetic information contributes to proper gene expression and development, and can be transmitted not only through mitotic divisions but also from parents to progeny. We investigated the roles in epigenetic inheritance of MES-4 and MET-1, the two Caenorhabditis elegans enzymes that methylate H3K36 (histone H3 Lys 36). Mass spectrometry analysis confirmed immunostaining results showing that both MES-4 and MET-1 catalyze H3K36me3. In the adult germline, MES-4 is enriched in the distal mitotic zone and MET-1 is enriched in the meiotic pachytene zone. Embryos inherit H3K36me3-marked chromosomes from both the oocyte and sperm, and a maternal load of MES-4 and MET-1 Maternal MES-4 quickly associates with sperm chromosomes; that association requires that the sperm chromosomes bear H3K36me3, suggesting that MES-4 is recruited to chromosomes by preexisting H3K36me3. In embryos that inherit H3K36me3-positive oocyte chromosomes and H3K36me3-negative sperm chromosomes, MES-4 and H3K36me3 are maintained on only a subset of chromosomes until at least the 32-cell stage, likely because MES-4 propagates H3K36me3 on regions of the genome with preexisting H3K36me3. In embryos lacking MES-4, H3K36me3 levels on chromosomes drop precipitously postfertilization. In contrast to the relatively high levels of MES-4 in early-stage embryos, MET-1 levels are low at early stages and start increasing by the ∼26-cell stage, consistent with expression from the zygotic genome. Our findings support the model that MET-1 mediates transcription-coupled H3K36me3 in the parental germline and transcriptionally active embryos, and that MES-4 transmits an epigenetic memory of H3K36me3 across generations and through early embryo cell divisions by maintaining inherited patterns of H3K36me3.

Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.

Germ cell tumors (GCTs) are a heterogeneous group of tumors occurring in gonadal and extragonadal locations. GCTs are hypothesized to arise from primordial germ cells (PGCs), which fail to differentiate. One recently identified susceptibility loci for human GCT is PR (PRDI-BF1 and RIZ) domain proteins 14 (PRDM14). PRDM14 is expressed in early primate PGCs and is repressed as PGCs differentiate. To examine PRDM14 in human GCTs we profiled human GCT cell lines and patient samples and discovered that PRDM14 is expressed in embryonal carcinoma cell lines, embryonal carcinomas, seminomas, intracranial germinomas and yolk sac tumors, but is not expressed in teratomas. To model constitutive overexpression in human PGCs, we generated PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs) and discovered that elevated expression of PRDM14 does not block early PGC formation. Instead, we show that elevated PRDM14 in PGCLCs causes proliferation and differentiation defects in the germline.

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFβ and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.

Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. Here we show that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, we show that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a (HP1a; encoded by Su(var)205). Furthermore, we show that this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. Our findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk -/- Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk -/- mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk +/+ and Mertk -/- mice. Prior to the onset of photoreceptor degeneration, Mertk -/- mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.