Two miRNA clusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), are involved in the regulation of spermatogonial differentiation in mice

Increasing evidence indicates that microRNAs (miRNAs) may be critical players in spermatogenesis. The miRNA expression profiles of THY1(+)-enriched undifferentiated spermatogonia were characterized, and members of Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) clusters are significantly downregulated during retinoic acid-induced spermatogonial differentiation, both in vitro and in vivo. The repression of microRNA clusters Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3) by retinoic acid in turn potentially upregulates the expression of Bim, Kit, Socs3, and Stat3. The male germ cell-specific Mir-17-92 (Mirc1) knockout mice exhibit small testes, a lower number of epididymal sperm, and mild defect in spermatogenesis. Absence of Mir-17-92 (Mirc1) in male germ cells dramatically increases expression of Mir-106b-25 (Mirc3) cluster miRNAs in the germ cells. These results suggest that Mir-17-92 (Mirc1) cluster and Mir-106b-25 (Mirc3) cluster miRNAs possibly functionally cooperate in regulating spermatogonial development.     

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.     

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.     

Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice

Self-renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR-486-5p enriched in Sertoli cell and Sertoli cell-derived exosomes. The exosomes mediate the transfer of miR-486-5p from Sertoli cells to SSCs. Exosomes release miR-486-5p, thus up-regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR-486-5p. Overexpression of miR-486-5p in SSCs down-regulates PTEN expression, which up-regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome-mediated transfer of miR-486-5p inhibits differentiation of SSC. Our findings demonstrate that miR-486-5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.     

Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro

In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6–8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10⁻⁷ mol l⁻¹ of RA effectively induced the SSCs into haploid male germ cells in vitro.     

Expression of the ubiquitin proteasome system in neonatal rat gonocytes and spermatogonia: role in gonocyte differentiation

The ubiquitin proteasome system (UPS) consists of a cascade of enzymatic reactions leading to the ubiquitination of proteins, with consequent degradation or altered functions of the proteins. Alterations in UPS genes have been associated with male infertility, suggesting the role of UPS in spermatogenesis. In the present study, we questioned whether UPS is involved in extensive remodeling and functional changes occurring during the differentiation of neonatal testicular gonocytes to spermatogonia, a step critical for the establishment of the spermatogonial stem cell population. We found that addition of the proteasome inhibitor lactacystin to isolated gonocytes inhibited their retinoic acid-induced differentiation in a dose-dependent manner, blocking the induction of the spermatogonial gene markers Stra8 and Dazl. We then compared the UPS gene expression profiles of Postnatal Day (PND) 3 gonocytes and PND8 spermatogonia, using gene expression arrays and quantitative real-time PCR analyses. We identified 205 UPS genes, including 91 genes expressed at relatively high levels. From those, 28 genes were differentially expressed between gonocytes and spermatogonia. While ubiquitin-activating enzymes and ligases showed higher expression in gonocytes, most ubiquitin conjugating and deubiquitinating enzymes were expressed at higher levels in spermatogonia. Concomitant with the induction of spermatogonial gene markers, retinoic acid altered the expression of many UPS genes, suggesting that the UPS is remodeled during gonocyte differentiation. In conclusion, these studies identified novel ubiquitin-related genes in gonocytes and spermatogonia and revealed that proteasome function is involved in gonocyte differentiation. Considering the multiple roles of the UPS, it will be important to determine which UPS genes direct substrates to the proteasome and which are involved in proteasome-independent functions in gonocytes and to identify their target proteins.     

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0–G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells.     

Spermatogenesis in retinol-deficient rats maintained on retinoic acid.

Rats maintained on a diet deficient in retinol and retinoic acid were given a diet containing retinoic acid for 21-29 days after the start of weight loss. The testes of four of these rats were studied. Spermatogonia of all types were observed, though in lower numbers than in controls, and their mitotic activity was normal. Normal preleptotene spermatocytes were encountered, but no normal spermatocytes in further stages of development were seen. Pale cells that appeared to be in prophase were observed. It was concluded that, in retinol-deficient rats maintained on retinoic acid, the spermatogonial population is qualitatively normal, but quantitatively subnormal, while spermatocyte development is qualitatively and quantitatively abnormal. No evidence of spermatogonial arrest or any other form of synchronization was found in testes of these rats, but when the remaining rats were injected with retinol, the seminiferous epithelium did show stage synchronization at 36 and 128 days after the injection.     

Importance of cell-aggregation during induction of neural differentiation in PCC-7 embryonal carcinoma cells.

The importance of cell-aggregation during retinoic acid-induced neural differentiation of embryonal carcinoma cells was studied on the PCC-7 cell line. These cells were chosen as they display low tendency for spontaneous aggregation, and they develop preferentially to neurons upon induced in vitro differentiation. Forced aggregation of these cells, in the absence of retinoic acid, did not result in development of neuron- or glial-like cells. Application of retinoic acid prior to or after the cell-aggregation did not result in neural tissue-like differentiation, either. Irreversible induction of neural development was achieved if cell-aggregation and retinonic acid acted simultaneously, and for a period longer than 48 h. Retinoic acid, on the other hand, was found to be toxic on non-aggregated PCC-7 cells. Our data suggest that cell to cell contacts alter the response of these cells to retinoic acid, and their close apposition is a prerequisite for the retinoic acid-induced neural differentiation.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.     

Bone morphogenetic protein signaling in articular chondrocyte differentiation

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.