Testicular Architecture Is Critical for Mediation of Retinoic Acid Responsiveness by Undifferentiated Spermatogonial Subtypes in the Mouse.
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SUBMITTER: Lord T
PROVIDER: S-EPMC5830974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the testis, a subset of spermatogonia retains stem cell potential, while others differentiate to eventually become spermatozoa. This delicate balance must be maintained, as defects can result in testicular cancer or infertility. Currently, little is known about the gene products and signaling pathways directing these critical cell fate decisions. Retinoic acid (RA) is a requisite driver of spermatogonial differentiation and entry into meiosis, yet the mechanisms activated downstream are undefined. Here, we determined a requirement for RA in the expression of KIT, a receptor tyrosine kinase essential for spermatogonial differentiation. We found that RA signaling utilized the PI3K/AKT/mTOR signaling pathway to induce the efficient translation of mRNAs for Kit, which are present but not translated in undifferentiated spermatogonia. Our findings provide an important molecular link between a morphogen (RA) and the expression of KIT protein, which together direct the differentiation of spermatogonia throughout the male reproductive lifespan.
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Project description:BackgroundCadherins play a pivotal role in facilitating intercellular interactions between spermatogonial progenitor cells (SPCs) and their surrounding microenvironment. Specifically, E-cadherin serves as a cellular marker of SPCs in many species. Depletion of E-cadherin in mouse SPCs showed no obvious effect on SPCs homing and spermatogenesis.ResultsHere, we investigated the regulatory role of E-cadherin in regulating SPCs fate. Specific deletion of E-cadherin in germ cells was shown to promote SPCs differentiation, evidencing by reduced PLZF+ population and increased c-Kit+ population in mouse testes. E-cadherin loss down-regulated the expression level of β-catenin, leading to the reduced β-catenin in nuclear localization for transcriptional activity. Remarkably, increasing expression level of Cadherin-22 (CDH22) appeared specifically after E-cadherin deletion, indicating CDH22 played a synergistic effect with E-cadherin in SPCs. By searching for the binding partners of β-catenin, Lymphoid enhancer-binding factor 1 (LEF1), T-cell factor (TCF3), histone deacetylase 4 (HDAC4) and signal transducer and activator 3 (STAT3) were identified as suppressors of SPCs differentiation by regulating acetylation of differentiation genes with PLZF.ConclusionsTwo surface markers of SPCs, E-cadherin and Cadherin-22, synergically maintain the undifferentiation of SPCs via the pivotal intermediate molecule β-catenin. LEF1, TCF3, STAT3 and HDAC4 were identified as co-regulatory factors of β-catenin in regulation of SPC fate. These observations revealed a novel regulatory pattern of cadherins on SPCs fate.
| S-EPMC9434974 | biostudies-literature
Project description:ObjectiveApplications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells.Materials and methodsIn this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson's trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC.ResultsHaematoxylin-eosin (H and E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This confirmed that our decellularisation protocol was efficient. Masson's trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules.ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.
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Project description:Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.
| S-EPMC4884469 | biostudies-literature
Project description:Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.
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Project description:Retinoic acid (RA) directs the sequential, but distinct, programs of spermatogonial differentiation and meiotic differentiation that are both essential for the generation of functional spermatozoa. These processes are functionally and temporally decoupled, as they occur in distinct cell types that arise over a week apart, both in the neonatal and adult testis. However, our understanding is limited in terms of what cellular and molecular changes occur downstream of RA exposure that prepare differentiating spermatogonia for meiotic initiation. In this review, we describe the process of spermatogonial differentiation and summarize the current state of knowledge regarding RA signaling in spermatogonia.
| S-EPMC4809555 | biostudies-literature
Project description:In the mammalian testis, sustained spermatogenesis relies on spermatogonial stem cells (SSCs); their progeny either remain as stem cells (self-renewal) or proliferate and differentiate to enter meiosis in response to retinoic acid (RA). Here, we sought to uncover elusive mechanisms regulating a key switch fundamental to spermatogonial fate: the capacity of spermatogonia to respond to RA. Using the developing mouse testis as a model, we found that spermatogonia and precursor prospermatogonia exhibit a heterogeneous capacity to respond to RA with at least two underlying causes. First, progenitor spermatogonia are prevented from responding to RA by catabolic activity of cytochrome P450 family 26 enzymes. Second, a smaller subset of undifferentiated spermatogonia enriched for SSCs exhibit catabolism-independent RA insensitivity. Moreover, for the first time, we observed that precursor prospermatogonia are heterogeneous and comprise subpopulations that exhibit the same differential RA responsiveness found in neonatal spermatogonia. We propose a novel model by which mammalian prospermatogonial and spermatogonial fates are regulated by their intrinsic capacity to respond (or not) to the differentiation signal provided by RA before, and concurrent with, the initiation of spermatogenesis.
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Project description:Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.
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Project description:Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3' untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.
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Project description:Cancer therapy and conditioning treatments of non-malignant diseases affect spermatogonial function and may lead to male infertility. Data on the molecular properties of spermatogonia and the influence of disease and/or treatment on spermatogonial subpopulations remain limited. Here, we assessed if the density and percentage of spermatogonial subpopulation changes during development (n = 13) and due to disease and/or treatment (n = 18) in tissues stored in fertility preservation programs, using markers for spermatogonia (MAGEA4), undifferentiated spermatogonia (UTF1), proliferation (PCNA), and global DNA methylation (5mC). Throughout normal prepubertal testicular development, only the density of 5mC-positive spermatogonia significantly increased with age. In comparison, patients affected by disease and/or treatment showed a reduced density of UTF1-, PCNA- and 5mC-positive spermatogonia, whereas the percentage of spermatogonial subpopulations remained unchanged. As an exception, sickle cell disease patients treated with hydroxyurea displayed a reduction in both density and percentage of 5mC- positive spermatogonia. Our results demonstrate that, in general, a reduction in spermatogonial density does not alter the percentages of undifferentiated and proliferating spermatogonia, nor the establishment of global methylation. However, in sickle cell disease patients', establishment of spermatogonial DNA methylation is impaired, which may be of importance for the potential use of this tissues in fertility preservation programs.
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