Chromatin remodelers HELLS, WDHD1 and BAZ1A are dynamically expressed during mouse spermatogenesis.

In brief: Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER. Abstract: Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.

DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis.

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

Molecular regulation of spermatogonial stem cell renewal and differentiation.

The intricate molecular and cellular interactions between spermatogonial stem cells (SSCs) and their cognate niche form the basis for life-long sperm production. To maintain long-term fertility and sustain sufficiently high levels of spermatogenesis, a delicate balance needs to prevail between the different niche factors that control cell fate decisions of SSCs by promoting self-renewal, differentiation priming or spermatogenic commitment of undifferentiated spermatogonia (Aundiff). Previously the SSC niche was thought to be formed primarily by Sertoli cells. However, recent research has indicated that many distinct cell types within the testis contribute to the SSC niche including most somatic cell populations and differentiating germ cells. Moreover, postnatal testis development involves maturation of somatic supporting cell populations and onset of cyclic function of the seminiferous epithelium. The stochastic and flexible behavior of Aundiff further complicates the definition of the SSC niche. Unlike in invertebrate species, providing a simple anatomical description of the SSC niche in the mouse is therefore challenging. Rather, the niche needs to be understood as a dynamic system that is able to serve the long-term reproductive function and maintenance of fertility both under steady-state and during development plus regeneration. Recent data from us and others have also shown that Aundiff reversibly transition between differentiation-primed and self-renewing states based on availability of niche-derived cues. This review focuses on defining the current understanding of the SSC niche and the elements involved in its regulation.

Distinct germline progenitor subsets defined through Tsc2-mTORC1 signaling.

Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional heterogeneity existing within this self-renewing population is poorly understood. Here, we define distinct subsets of undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation of mTORC1, a key growth-promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 throughout the SPC pool using Vasa-Cre promotes differentiation at the expense of self-renewal and leads to germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8-Cre did not compromise SPC function. SPC activity also appeared unaffected by Amh-Cre-mediated Tsc2 deletion within somatic cells of the niche. Importantly, we find that differentiation-prone SPCs have elevated mTORC1 activity when compared to SPCs with high self-renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with mTORC1-active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and defines phenotypically distinct SPC subpopulations with varying propensities for self-renewal and differentiation.

Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies.

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

Mechanisms of thyrotropin receptor-mediated phenotype variability deciphered by gene mutations and M453T-knockin model.

The clinical spectrum of thyrotropin receptor-mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T-knockin model revealed that the phenotype was dependent on the mutation's signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis.

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.

Retinoblastoma-E2F Transcription Factor Interplay Is Essential for Testicular Development and Male Fertility.

The retinoblastoma (RB) protein family members (pRB, p107 and p130) are key regulators of cell cycle progression, but also play crucial roles in apoptosis, and stem cell self-renewal and differentiation. RB proteins exert their effects through binding to E2F transcription factors, which are essential developmental and physiological regulators of tissue and organ homeostasis. According to the canonical view, phosphorylation of RB results in release of E2Fs and induction of genes needed for progress of the cell cycle. However, there are eight members in the E2F transcription factor family with both activator (E2F1-3a) and repressor (E2F3b-E2F8) roles, highlighting the functional diversity of RB-E2F pathway. In this review article we summarize the data showing that RB-E2F interaction is a key cell-autonomous mechanism responsible for establishment and maintenance of lifelong male fertility. We also review the expression pattern of RB proteins and E2F transcription factors in the testis and male germ cells. The available evidence supports that RB and E2F family members are widely and dynamically expressed in the testis, and they are known to have versatile roles during spermatogenesis. Knowledge of the function and significance of RB-E2F interplay for testicular development and spermatogenesis comes primarily from gene knock-out (KO) studies. Several studies conducted in Sertoli cell-specific pRB-KO mice have demonstrated that pRB-mediated inhibition of E2F3 is essential for Sertoli cell functional maturation and cell cycle exit, highlighting that RB-E2F interaction in Sertoli cells is paramount to male fertility. Similarly, ablation of either pRB or E2F1 in the germline results in progressive testicular atrophy due to germline stem cell (GSC) depletion, emphasizing the importance of proper RB-E2F interplay for germline maintenance and lifelong sperm production. In summary, while balanced RB-E2F interplay is essential for cell-autonomous maintenance of GSCs and, the pRB-E2F3 system in Sertoli cells is critical for providing GSC niche thus laying the basis for spermatogenesis.

STAGETOOL, a Novel Automated Approach for Mouse Testis Histological Analysis.

Spermatogenesis is a complex differentiation process that takes place in the seminiferous tubules. A specific organization of spermatogenic cells within the seminiferous epithelium enables a synchronous progress of germ cells at certain steps of differentiation on the spermatogenic pathway. This can be observed in testis cross-sections where seminiferous tubules can be classified into distinct stages of constant cellular composition (12 stages in the mouse). For a detailed analysis of spermatogenesis, these stages have to be individually observed from testis cross-sections. However, the recognition of stages requires special training and expertise. Furthermore, the manual scoring is laborious considering the high number of tubule cross-sections that have to be analyzed. To facilitate the analysis of spermatogenesis, we have developed a convolutional deep neural network-based approach named "STAGETOOL." STAGETOOL analyses histological images of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)-stained mouse testis cross-sections at ×400 magnification, and very accurately classifies tubule cross-sections into 5 stage classes and cells into 9 categories. STAGETOOL classification accuracy for stage classes of seminiferous tubules of a whole-testis cross-section is 99.1%. For cellular level analysis the F1 score for 9 seminiferous epithelial cell types ranges from 0.80 to 0.98. Furthermore, we show that STAGETOOL can be applied for the analysis of knockout mouse models with spermatogenic defects, as well as for automated profiling of protein expression patterns. STAGETOOL is the first fluorescent labeling-based automated method for mouse testis histological analysis that enables both stage and cell-type recognition. While STAGETOOL qualitatively parallels an experienced human histologist, it outperforms humans time-wise, therefore representing a major advancement in male reproductive biology research.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Hedgehog signalling promotes germ cell survival in the rat testis.

Hedgehog (Hh) signalling has a crucial role in testis development. Sertoli cell-derived desert hedgehog (DHH) guides the formation of testis cords and differentiation of foetal-type Leydig cells. Dhh mutant mice are infertile due to a block in germ cell differentiation, hypogonadism and hypoandrogenism. Hh signalling pathway components are also expressed in postnatal testis. In the rat testis the transcription factor of the Hh pathway, glioma-associated oncogene homologue (GLI1), is expressed by a wide variety of germ cells. This suggests that Hh signalling is involved in spermatogenesis at many different levels. Our data show that canonical Hh signalling is turned off in early condensing spermatids that strongly express the negative regulator of the pathway, suppressor of fused (SUFU). Most of the Hh pathway specific mRNAs display the highest values in stages II-VI of the rat seminiferous epithelial cycle. The key endocrine regulator of germ cell differentiation, FSH, down-regulates Dhh mRNA levels in vitro. Hh signalling inhibition in vitro leads to massive apoptosis of germ cells. In prepubertal rat testis imatinib mesylate-induced inhibition of tyrosine kinases impinges on Dhh transcript levels and Hh signalling. Our data indicate that Hh signalling is part of the paracrine signalling network in the rat testis. It promotes the survival of germ cells and is suppressed by FSH.

SOX3 promotes generation of committed spermatogonia in postnatal mouse testes.

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

Generation, localization and functions of macrophages during the development of testis.

In the testis, interstitial macrophages are thought to be derived from the yolk sac during fetal development, and later replaced by bone marrow-derived macrophages. By contrast, the peritubular macrophages have been reported to emerge first in the postnatal testis and solely represent descendants of bone marrow-derived monocytes. Here, we define new monocyte and macrophage types in the fetal and postnatal testis using high-dimensional single-cell analyses. Our results show that interstitial macrophages have a dominant contribution from fetal liver-derived precursors, while peritubular macrophages are generated already at birth from embryonic precursors. We find that bone marrow-derived monocytes do not substantially contribute to the replenishment of the testicular macrophage pool even after systemic macrophage depletion. The presence of macrophages prenatally, but not postnatally, is necessary for normal spermatogenesis. Our multifaceted data thus challenge the current paradigms in testicular macrophage biology by delineating their differentiation, homeostasis and functions.

Hypogonadism and Cryptorchidism.

Congenital cryptorchidism (undescended testis) is one of the most common congenital urogenital malformations in boys. Prevalence of cryptorchidism at birth among boys born with normal birth weight ranges from 1.8 to 8.4%. Cryptorchidism is associated with a risk of low semen quality and an increased risk of testicular germ cell tumors. Testicular hormones, androgens and insulin-like peptide 3 (INSL3), have an essential role in the process of testicular descent from intra-abdominal position into the scrotum in fetal life. This explains the increased prevalence of cryptorchidism among boys with diseases or syndromes associated with congenitally decreased secretion or action of androgens, such as patients with congenital hypogonadism and partial androgen insensitivity syndrome. There is evidence to support that cryptorchidism is associated with decreased testicular hormone production later in life. It has been shown that cryptorchidism impairs long-term Sertoli cell function, but may also affect Leydig cells. Germ cell loss taking place in the cryptorchid testis is proportional to the duration of the condition, and therefore early orchiopexy to bring the testis into the scrotum is the standard treatment. However, the evidence for benefits of early orchiopexy for testicular endocrine function is controversial. The hormonal treatments using human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone (GnRH) to induce testicular descent have low success rates, and therefore they are not recommended by the current guidelines for management of cryptorchidism. However, more research is needed to assess the effects of hormonal treatments during infancy on future male reproductive health.

Testis Development.

Production of sperm and androgens is the main function of the testis. This depends on normal development of both testicular somatic cells and germ cells. A genetic program initiated from the Y chromosome gene sex-determining region Y (SRY) directs somatic cell specification to Sertoli cells that orchestrate further development. They first guide fetal germ cell differentiation toward spermatogenic destiny and then take care of the full service to spermatogenic cells during spermatogenesis. The number of Sertoli cells sets the limits of sperm production. Leydig cells secrete androgens that determine masculine development. Testis development does not depend on germ cells; that is, testicular somatic cells also develop in the absence of germ cells, and the testis can produce testosterone normally to induce full masculinization in these men. In contrast, spermatogenic cell development is totally dependent on somatic cells. We herein review germ cell differentiation from primordial germ cells to spermatogonia and development of the supporting somatic cells. Testicular descent to scrota is necessary for normal spermatogenesis, and cryptorchidism is the most common male birth defect. This is a mild form of a disorder of sex differentiation. Multiple genetic reasons for more severe forms of disorders of sex differentiation have been revealed during the last decades, and these are described along with the description of molecular regulation of testis development.

Transcription Factor USF1 Is Required for Maintenance of Germline Stem Cells in Male Mice.

A prerequisite for lifelong sperm production is that spermatogonial stem cells (SSCs) balance self-renewal and differentiation, yet factors required for this balance remain largely undefined. Using mouse genetics, we now demonstrate that the ubiquitously expressed transcription factor upstream stimulatory factor (USF)1 is critical for the maintenance of SSCs. We show that USF1 is not only detected in Sertoli cells as previously reported, but also in SSCs. Usf1-deficient mice display progressive spermatogenic decline as a result of age-dependent loss of SSCs. According to our data, the germ cell defect in Usf1-/- mice cannot be attributed to impairment of Sertoli cell development, maturation, or function, but instead is likely due to an inability of SSCs to maintain a quiescent state. SSCs of Usf1-/- mice undergo continuous proliferation, which provides an explanation for their age-dependent depletion. The proliferation-coupled exhaustion of SSCs in turn results in progressive degeneration of the seminiferous epithelium, gradual decrease in sperm production, and testicular atrophy. We conclude that the general transcription factor USF1 is indispensable for the proper maintenance of mammalian spermatogenesis.

Identification of dynamic undifferentiated cell states within the male germline.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.