The people behind the papers - Tiffany Roach and Kari Lenhart.

Germ stem cells in Drosophila reside within a specialized stem cell niche, but the effects of stress on these stem cell populations have been elusive. In a new study, Roach and Lenhart show that repeated mating stress induces reversible changes in the germ stem cell niche. To know more about their work, we spoke to first author, Tiffany Roach, and corresponding author, Kari Lenhart, Principal Investigator at Drexel University in Philadelphia, USA.

Mating-induced Ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis.

Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, we find that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. We find that GSC abscission failure is caused by increased Ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females, but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population.

Tumor espermatocítico variante anaplásica sincrónico bilateral: reporte de una neoplasia infrecuente / Bilateral synchronous anaplastic variant spermatocytic tumor: Report of a rare neoplasm

El tumor espermatocítico es una neoplasia testicular de células germinales, muy infrecuente, que representa menos del 1% de los cánceres testiculares. Afecta generalmente a hombres mayores con una edad media de 53,6 años (rango 19-92 años). El tumor espermatocítico se clasifica dentro del grupo de tumores germinales no relacionados con la neoplasia de células germinales in situ. Muestra características clinicopatológicas diferentes del seminoma clásico y no se considera una variante de este último. Debido a una superposición morfológica con el seminoma clásico, en el pasado se denominó «seminoma espermatocítico». La variante anaplásica del tumor espermatocítico es excepcional, se han descrito pocos casos en la literatura, presenta un inicio más temprano en comparación con el tumor espermatocítico y un buen comportamiento a pesar de mostrar patrones histológicos similares al seminoma clásico. Presentamos el segundo caso de tumor espermatocítico anaplásico sincrónico bilateral, en un paciente joven tratado con orquiectomía y quimioterapia. (AU)

Spermatocytic tumor is a very rare germ cell testicular neoplasm that accounts for less than 1% of testicular cancers. It generally affects older men with a mean age of 53.6 years (range 19-92 years). Spermatocytic tumor is classified within the group of germ cell tumors not related to germ cell neoplasia in situ. It presents clinicopathological characteristics different from classic seminoma and is not considered a variant of the latter. Due to a morphologic overlap with classical seminoma, it was called “sperm cell seminoma” in the past. The anaplastic variant of spermatocytic tumor is exceptional, few cases have been described in the literature, it presents an earlier onset compared to spermatocytic tumor and a benign behavior despite showing histological patterns similar to classic seminoma. We present the second case of bilateral synchronous anaplastic spermatocytic tumor, in a young patient treated with orchiectomy and chemotherapy. (AU)

Innovations in poultry reproduction using cryopreserved avian germ cells.

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals.

Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Gametes of semelparous salmon are repeatedly produced by surrogate rainbow trout.

Most Pacific salmon species grow in the ocean, return to their native rivers to reproduce, and then die (semelparous type). However, rainbow trout survive after spawning and reproduce repeatedly until the end of their lives (iteroparous type). Little is known about how germline stem cells behave during gametogenesis in the two types of Pacific salmon. In this study, we show that all germline stem cells disappear after the first gametogenesis in Chinook and Kokanee salmon, whereas germline stem cells are maintained in rainbow trout. However, the germline stem cells of Chinook and Kokanee salmon transplanted into rainbow trout survive even after their spawning seasons and supply salmon gametes for multiple years. These results indicate that the behavior of the germline stem cells is mainly regulated by the somatic environment.

Type 1 diabetes impairs the activity of rat testicular somatic and germ cells through NRF2/NLRP3 pathway-mediated oxidative stress.

Background: It is well known that metabolic disorders, including type 1 diabetes (T1D), are often associated with reduced male fertility, mainly increasing oxidative stress and impairing the hypothalamus-pituitary-testis (HPT) axis, with consequently altered spermatogenesis and reduced sperm parameters. Herein, using a rat model of T1D obtained by treatment with streptozotocin (STZ), we analyzed several parameters of testicular activity. Methods: A total of 10 adult male Wistar rats were divided into two groups of five: control and T1D, obtained with a single intraperitoneal injection of STZ. After 3 months, the rats were anesthetized and sacrificed; one testis was stored at -80°C for biochemical analysis, and the other was fixed for histological and immunofluorescence analysis. Results: The data confirmed that T1D induced oxidative stress and, consequently, alterations in both testicular somatic and germ cells. This aspect was highlighted by enhanced apoptosis, altered steroidogenesis and Leydig cell maturity, and impaired spermatogenesis. In addition, the blood-testis barrier integrity was compromised, as shown by the reduced levels of structural proteins (N-cadherin, ZO-1, occludin, connexin 43, and VANGL2) and the phosphorylation status of regulative kinases (Src and FAK). Mechanistically, the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways was proven, particularly the reduced nuclear translocation of NRF2, affecting its ability to induce the transcription of genes encoding for antioxidant enzymes. Finally, the stimulation of testicular inflammation and pyroptosis was also confirmed, as highlighted by the increased levels of some markers, such as NF-κB and NLRP3. Conclusion: The combined data allowed us to confirm that T1D has detrimental effects on rat testicular activity. Moreover, a better comprehension of the molecular mechanisms underlying the association between metabolic disorders and male fertility could help to identify novel targets to prevent and treat fertility disorders related to T1D.

Germline-mediated immunoediting sculpts breast cancer subtypes and metastatic proclivity.

Tumors with the same diagnosis can have different molecular profiles and response to treatment. It remains unclear when and why these differences arise. Somatic genomic aberrations occur within the context of a highly variable germline genome. Interrogating 5870 breast cancer lesions, we demonstrated that germline-derived epitopes in recurrently amplified genes influence somatic evolution by mediating immunoediting. Individuals with a high germline-epitope burden in human epidermal growth factor receptor 2 (HER2/ERBB2) are less likely to develop HER2-positive breast cancer compared with other subtypes. The same holds true for recurrent amplicons defining three aggressive estrogen receptor (ER)-positive subgroups. Tumors that overcome such immune-mediated negative selection are more aggressive and demonstrate an "immune cold" phenotype. These data show that the germline genome plays a role in dictating somatic evolution.

RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.

Germline Cas9 promoters with improved performance for homing gene drive.

Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives convert wild type alleles into drive alleles in heterozygotes with Cas9 and gRNA. It is thus desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we test eleven Drosophila melanogaster germline promoters. Some achieve higher drive conversion efficiency with minimal embryo resistance, but none completely avoid somatic expression. However, such somatic expression often does not carry detectable fitness costs for a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Supporting a 4-gRNA suppression drive, one promoter leads to a low drive equilibrium frequency due to fitness costs from somatic expression, but the other outperforms nanos, resulting in successful suppression of the cage population. Overall, these Cas9 promoters hold advantages for homing drives in Drosophila species and may possess valuable homologs in other organisms.

The matrisome landscape controlling in vivo germ cell fates.

The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.

piRNA-Guided Transposon Silencing and Response to Stress in Drosophila Germline.

Transposons are integral genome constituents that can be domesticated for host functions, but they also represent a significant threat to genome stability. Transposon silencing is especially critical in the germline, which is dedicated to transmitting inherited genetic material. The small Piwi-interacting RNAs (piRNAs) have a deeply conserved function in transposon silencing in the germline. piRNA biogenesis and function are particularly well understood in Drosophila melanogaster, but some fundamental mechanisms remain elusive and there is growing evidence that the pathway is regulated in response to genotoxic and environmental stress. Here, we review transposon regulation by piRNAs and the piRNA pathway regulation in response to stress, focusing on the Drosophila female germline.

Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Exploration of cell-cell interactions and the notch signaling pathway in the gonadal niche of Crassostrea gigas.

The Notch signaling pathway plays a pivotal role in governing cell fate determinations within the gonadal niche. This study provides an extensive elucidation of the male and female gonadal niches within Crassostrea gigas. Examination via transmission electron microscopy revealed the presence of desmosome-like connection not only between germ cells and niche cells but also among adjacent niche cells within the oyster gonad. Transcriptomic analysis identified several putative Notch pathway components, including CgJAG1, CgNOTCH1, CgSuh, and CgHey1. Phylogenetic analysis indicated a close evolutionary relationship between CgJAG1, CgNOTCH1, and CgHey1 and Notch members present in Drosophila. Expression profiling results indicated a notable abundance of CgHey1 in the gonads, while CgJAG1 and CgNOTCH1 displayed distinct expression patterns associated with sexual dimorphism. In situ hybridization findings corroborated the predominant expression of CgJAG1 in male niche cells, while CgNOTCH1 was expressed in both male and female germ cells, as well as female niche cells. These findings demonstrate the important role of the Notch signaling pathway in the gonadal niche of oysters.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Uptake of Risk-Reducing Measures, Cascade Testing, and Related Challenges Among Carriers of Breast Cancer-Associated Germline Pathogenic Variants in Mexico.

PURPOSE: Genetic cancer risk assessment (GCRA) provides pathogenic variant (PV) carriers with the invaluable opportunity to undertake timely cancer risk-reducing (RR) measures and initiate cascade testing (CT). This study describes the uptake of these strategies and the related barriers among breast cancer-associated germline PV carriers in Mexico. METHODS: Carriers who were at least 6 months after disclosure of genetic test results at two GCRA referral centers were invited to answer a survey assessing sociodemographic characteristics, awareness of their carrier status and its implications, uptake of RR measures according to international guidelines by PV, CT initiation, and associated challenges. RESULTS: Of the eligible carriers, 246/384 (64%) answered the survey (median age: 44 years). Most were female (88%), married/in domestic partnership (66%), and had personal breast/ovarian cancer history (61%). PVs included BRCA1/2 (75%), CHEK2 (10%), PALB2 (5%), ATM (5%), NF1 (2%), RAD51C (2%), PTEN (1%), and TP53 (1%). Most (87%) participants were aware of their carrier status. When recommended, 37% underwent RR bilateral mastectomy, 48% RR oophorectomy, 70% annual mammogram, and 20% breast magnetic resonance imaging. Challenges hindering the uptake of RR measures included financial limitations (67%), lack of recommendation by their physician (35%), and fear (24%). Nearly all (98%) claimed sharing their results with their relatives. CT was initiated in 63% of families and was associated with carriers being married/in domestic partnership (P = .04) and believing GCRA was useful (P < .001). CONCLUSION: Despite the resource-constrained setting, relevant rates of RR measures and CT were observed. Targeted interventions to reduce out-of-pocket expenses and improve patient-physician communication and patients' understanding on carrier status are warranted to enhance the overall benefit of GCRA and ultimately improve the provision of patient-centered care to both carriers and their at-risk relatives.

Smaug regulates germ plasm assembly and primordial germ cell number in Drosophila embryos.

During Drosophila oogenesis, the Oskar (OSK) RNA binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here, we identify mechanisms that subsequently regulate germ plasm assembly in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk messenger RNA (mRNA) as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNA results in excess translation of these transcripts in the germ plasm, accumulation of excess germ plasm, and budding of excess primordial germ cells (PGCs). Therefore, SMG triggers a posttranscriptional regulatory pathway that attenuates the amount of germ plasm in embryos to modulate the number of PGCs.