Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Dissecting the dynamic cellular transcriptional atlas of adult teleost testis development throughout the annual reproductive cycle.

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species.

TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

Retinoic acid is dispensable for meiotic initiation but required for spermiogenesis in the mammalian testis.

Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell differentiation and meiotic initiation are temporally inseparable. In the postnatal testis, these events are separated by more than 1 week. Exploiting this difference, we discovered that, although RA is required for spermatogonial differentiation, it is dispensable for the subsequent initiation, progression and completion of meiosis. Indeed, in the absence of RA, the meiotic transcriptome program in both differentiating spermatogonia and spermatocytes entering meiosis was largely unaffected. Instead, transcripts encoding factors required during spermiogenesis were aberrant during preleptonema, and the subsequent spermatid morphogenesis program was disrupted such that no sperm were produced. Taken together, these data reveal a RA-independent model for male meiotic initiation.

Biased precursor ingression underlies the center-to-pole pattern of male sex determination in mouse.

During mammalian development, gonadal sex determination results from the commitment of bipotential supporting cells to Sertoli or granulosa cell fates. Typically, this decision is coordinated across the gonad to ensure commitment to a single organ fate. When unified commitment fails in an XY mouse, an ovotestis forms in which supporting cells in the center of the gonad typically develop as Sertoli cells, while supporting cells in the poles develop as granulosa cells. This central bias for Sertoli cell fate was thought to result from the initial expression of the drivers of Sertoli cell fate, SRY and/or SOX9, in the central domain, followed by paracrine expansion to the poles. However, we show here that the earliest cells expressing SRY and SOX9 are widely distributed across the gonad. In addition, Sertoli cell fate does not spread among supporting cells through paracrine relay. Instead, we uncover a center-biased pattern of supporting cell precursor ingression that occurs in both sexes and results in increased supporting cell density in the central domain. Our findings prompt a new model of gonad patterning in which a density-dependent organizing principle dominates Sertoli cell fate stabilization.

Sertoli cells are the source of stem cell factor for spermatogenesis.

Several cell types have been proposed to create the required microenvironment for spermatogenesis. However, expression patterns of the key growth factors produced by these somatic cells have not been systematically studied and no such factor has been conditionally deleted from its primary source(s), raising the question of which cell type(s) are the physiological sources of these growth factors. Here, using single-cell RNA sequencing and a series of fluorescent reporter mice, we found that stem cell factor (Scf), one of the essential growth factors for spermatogenesis, was broadly expressed in testicular stromal cells, including Sertoli, endothelial, Leydig, smooth muscle and Tcf21-CreER+ stromal cells. Both undifferentiated and differentiating spermatogonia were associated with Scf-expressing Sertoli cells in the seminiferous tubule. Conditional deletion of Scf from Sertoli cells, but not any other Scf-expressing cells, blocked the differentiation of spermatogonia, leading to complete male infertility. Conditional overexpression of Scf in Sertoli cells, but not endothelial cells, significantly increased spermatogenesis. Our data reveal the importance of anatomical localization for Sertoli cells in regulating spermatogenesis and that SCF produced specifically by Sertoli cells is essential for spermatogenesis.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell-death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a "pre-granulosa-like" identity, while other revert to a "supporting progenitor-like" cell identity, most of them being "intersex" because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

TrxT and dhd are dispensable for Drosophila brain development but essential for l(3)mbt brain tumour growth.

Expression of the Drosophila cancer-germline (CG), X-linked, head-to-head gene pair TrxT and dhd is normally germline-specific but becomes upregulated in brain tumours caused by mutation in l(3)mbt. Here, we show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable, taking into account that these two genes are never expressed together under normal conditions. We also show that TrxT, but not dhd, is crucial for the growth of l(3)mbt allografts, hence suggesting that the initial stages of tumour development and long-term tumour growth may depend on different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among CG genes that map to the X chromosome. Our results identify a first example of an X-linked, head-to-head CG gene pair in Drosophila, underpinning the potential of such CG genes, dispensable for normal development and homoeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors.

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete but abnormally activated in a wide variety of tumors. The CTA, Testis-specific serine kinase 6 (TSSK6), is essential for male fertility in mice. The functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse-free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage-independent growth, invasion, and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin-positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

Modeling mammalian spermatogonial differentiation and meiotic initiation in vitro.

In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

Loss of WNT4 in the gubernaculum causes unilateral cryptorchidism and fertility defects.

Undescended testis (UDT) affects 6% of male births. Despite surgical correction, some men with unilateral UDT may experience infertility with the contralateral descended testis (CDT) showing no A-dark spermatogonia. To improve our understanding of the etiology of infertility in UDT, we generated a novel murine model of left unilateral UDT. Gubernaculum-specific Wnt4 knockout (KO) mice (Wnt4-cKO) were generated using retinoic acid receptor ß2-cre mice and were found to have a smaller left-unilateral UDT. Wnt4-cKO mice with abdominal UDT had an increase in serum follicle-stimulating hormone and luteinizing hormone and an absence of germ cells in the undescended testicle. Wnt4-cKO mice with inguinal UDT had normal hormonal profiles, and 50% of these mice had no sperm in the left epididymis. Wnt4-cKO mice had fertility defects and produced 52% fewer litters and 78% fewer pups than control mice. Wnt4-cKO testes demonstrated increased expression of estrogen receptor α and SOX9, upregulation of female gonadal genes, and a decrease in male gonadal genes in both CDT and UDT. Several WNT4 variants were identified in boys with UDT. The presence of UDT and fertility defects in Wnt4-cKO mice highlights the crucial role of WNT4 in testicular development.

PBAF chromatin remodeler complexes that mediate meiotic transitions in mouse.

Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.

Casein kinase 1α regulates murine spermatogenesis via p53-Sox3 signaling.

Casein kinase 1α (CK1α), acting as one member of the ß-catenin degradation complex, negatively regulates the Wnt/ß-catenin signaling pathway. CK1α knockout usually causes both Wnt/ß-catenin and p53 activation. Our results demonstrated that conditional disruption of CK1α in spermatogonia impaired spermatogenesis and resulted in male mouse infertility. The progenitor cell population was dramatically decreased in CK1α conditional knockout (cKO) mice, while the proliferation of spermatogonial stem cells (SSCs) was not affected. Furthermore, our molecular analyses identified that CK1α loss was accompanied by nuclear stability of p53 protein in mouse spermatogonia, and dual-luciferase reporter and chromatin immunoprecipitation assays revealed that p53 directly targeted the Sox3 gene. In addition, the p53 inhibitor pifithrin α (PFTα) partially rescued the phenotype observed in cKO mice. Collectively, our data suggest that CK1α regulates spermatogenesis and male fertility through p53-Sox3 signaling, and they deepen our understanding of the regulatory mechanism underlying the male reproductive system.

Sex Determination in the Mammalian Germline.

Sexual reproduction crucially depends on the production of sperm in males and oocytes in females. Both types of gamete arise from the same precursor, the germ cells. We review the events that characterize the development of germ cells during fetal life as they commit to, and prepare for, oogenesis or spermatogenesis. In females, fetal germ cells enter meiosis, whereas in males they delay meiosis and instead lose pluripotency, activate an irreversible program of prospermatogonial differentiation, and temporarily cease dividing. Both pathways involve sex-specific molecular signals from the somatic cells of the developing gonads and a suite of intrinsic receptors, signal transducers, transcription factors, RNA stability factors, and epigenetic modulators that act in complex, interconnected positive and negative regulatory networks. Understanding these networks is important in the contexts of the etiology, diagnosis, and treatment of infertility and gonadal cancers, and in efforts to augment human and animal fertility using stem cell approaches.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Genomic analysis of spermatocytic tumors demonstrates recurrent molecular alterations in cases with malignant clinical behavior.

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

How germline genes promote malignancy in cancer cells.

Cancers often express hundreds of genes otherwise specific to germ cells, the germline/cancer (GC) genes. Here, we present and discuss the hypothesis that activation of a "germline program" promotes cancer cell malignancy. We do so by proposing four hallmark processes of the germline: meiosis, epigenetic plasticity, migration, and metabolic plasticity. Together, these hallmarks enable replicative immortality of germ cells as well as cancer cells. Especially meiotic genes are frequently expressed in cancer, implying that genes unique to meiosis may play a role in oncogenesis. Because GC genes are not expressed in healthy somatic tissues, they form an appealing source of specific treatment targets with limited side effects besides infertility. Although it is still unclear why germ cell specific genes are so abundantly expressed in cancer, from our hypothesis it follows that the germline's reproductive program is intrinsic to cancer development.

Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.