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Project description:Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3' UTRs of ~2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3' UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.
| S-EPMC7445011 | biostudies-literature
Project description:DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors
| PRJNA606263 | ENA
Project description:Although mitochondrial morphology is well-known for its role in cellular homeostasis, there is surprisingly little knowledge on whether mitochondrial remodeling is required for postnatal germ cell development. In this study, we investigated the functions of MFN1 and MFN2, two GTPases in mitochondrial fusion, during early spermatogenesis. We observed increased MFN expressions along with increased mitochondrial and endoplasmic reticulum (ER) activities during spermatogonial differentiation. Deletion of either Mfn led to DNA oxidation and apoptosis specifically in differentiating spermatogonia and spermatocytes, which in turn caused male infertility. We further found MFN2 regulated spermatogenesis by modulating both mitochondrial and ER functions, a mechanism distinct from that of MFN1. Defects of germ cell development in MFN2 mutants were corrected by MFN2 at either mitochondria or ER but not by MFN1. Our study thus reveals an essential requirement of MFN-mediated mitochondrial and ER coordination in spermatogenesis, providing critical insights into mitochondrial determinants of male fertility.
| S-EPMC7221103 | biostudies-literature
Project description:Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.
| S-EPMC10649044 | biostudies-literature
Project description:Expression of DAZ-like (DAZL) is a hallmark of vertebrate germ cells, and is essential for embryonic germ cell development and differentiation, yet the gametogenic function of DAZL has not been fully characterized and most of its in vivo direct targets remain unknown. We showed that postnatal stage-specific deletion of Dazl in mouse germ cells did not affect female fertility, but caused complete male sterility with gradual loss of spermatogonial stem cells, meiotic arrest and spermatid arrest. Using the genome-wide high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation and mass spectrometry approach, we found that DAZL bound to a large number of testicular mRNA transcripts (at least 3008) at the 3'-untranslated region and interacted with translation proteins including poly(A) binding protein. In the absence of DAZL, polysome-associated target transcripts, but not their total transcripts, were significantly decreased, resulting in a drastic reduction of an array of spermatogenic proteins and thus developmental arrest. Thus, DAZL is a master translational regulator essential for spermatogenesis.
| S-EPMC6660020 | biostudies-literature
Project description:Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.
| S-EPMC5736635 | biostudies-literature
Project description:Molecular markers of spermatogonia are necessary for studies on spermatogonial stem cells (SSCs) and improving our understanding of molecular and cellular biology of spermatogenesis. Although studies of germ cell surface marker have been extensively conducted in the testes of rodents, these markers have not been well studied in domestic animals. We aimed to determine the expression pattern of cluster of differentiation 14 (CD14) in developing porcine testes and cultured porcine SSCs (pSSCs), as well as its role in pSSC colony formation. Interestingly, expression of CD14 was observed in porcine testes with PGP9.5-positive undifferentiated spermatogonia at all developmental stages. In addition, in vitro cultured pSSCs expressed CD14 and showed successful colony formation, as determined by fluorescence-activated cell sorting and flow cytometry. PKH26 dye-stained CD14-positive cells transplants were performed into the testes of recipient mice, which were depleted of both testicular germ and somatic cells from immunodeficiency mice and were shown to colonise the recipient testes. Moreover, a colony-forming assay showed that the development of pSSC colonies was disrupted by a high concentration of lipopolysaccharide. These studies indicated that CD14 is surface marker of early spermatogonia in developing porcine testes and in pSSCs, suggesting a role for CD14 in porcine spermatogenesis.
| S-EPMC6620343 | biostudies-literature
Project description:Retinoic acid (RA) signaling is crucial for spermatogonial differentiation, which is a key step for spermatogenesis. We explored the mechanisms underlying spermatogonial differentiation by targeting expression of a dominant-negative mutant of retinoic acid receptor α (RARα) specifically to the germ cells of transgenic mice to subvert the activity of endogenous receptors. Here we show that: (1) inhibition of retinoid signaling in germ cells completely blocked spermatogonial differentiation identical to vitamin A-deficient (VAD) mice; (2) the blockage of spermatogonial differentiation by impaired retinoid signaling resulted from an arrest of entry of the undifferentiated spermatogonia into S phase; and (3) retinoid signaling regulated spermatogonial differentiation through controlling expression of its direct target genes, including replication-dependent core histone genes. Taken together, our results demonstrate that the action of retinoid signaling on spermatogonial differentiation in vivo is direct through the spermatogonia itself, and provide the first evidence that this is mediated by regulation of expression of replication-dependent core histone genes.
| S-EPMC4986167 | biostudies-literature
Project description:Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.
| S-EPMC6894055 | biostudies-literature
Project description:The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases.
| S-EPMC4754542 | biostudies-literature